DiVA portal1258091/FULLTEXT01.pdf · Två proteinkomplex står i fokus, BRG1 och BRM-innehållande...

Gene regulation by chromatin remodellingcomplexesSWI/SNF complex in mRNA processing and B-WICH complex inribosomal gene expressionAntoni Gañez Zapater

Academic dissertation for the Degree of Doctor of Philosophy in Cell Biology at StockholmUniversity to be publicly defended on Friday 7 December 2018 at 09.15 in Vivi Täckholmsalen(Q-salen), NPQ-huset, Svante Arrhenius väg 20.

AbstractThe aim of this project is to investigate the roles of chromatin remodelling complexes in gene regulation. It is focusedon two groups of chromatin complexes: the mammalian BRG1 and BRM SWI/SNF complexes and the ISWI-containingB-WICH complex.

Study 1 investigates the role of SWI/SNF complexes in alternative splicing. We show that the presence of the ATPasecore subunits Brg1 and Brm influence the alternative splicing outcome of a subset of genes. We show that Brg1 and Brminteract with several splicing related factors in the nascent RNA, and that the recruitment of some of these factors to theirtarget sites is regulated by the presence of Brg1 and Brm. We propose that SWI/SNF ATPases can modulate the interactionsof RNA binding factors to the nascent RNA and in that way alter alternative splicing outcome.

Study 2 focuses on SWI/SNF complexes and their influence on cleavage and polyadenylation of mRNA. We showthat Brg1 and Brm interact with subunits of the cleavage and polyadenylation complexes in the nascent mRNA. SWI/SNFcomplexes facilitate the recruitment of the cleavage and polyadenylation complex to the polyadenylation site in a subsetof genes, and this results in a more efficient cleavage and polyadenylation.

Study 3 shows that B-WICH is required for ribosome gene transcriptional activation upon glucose stimulation. WSTFand SNF2h, two of the B-WICH subunits, are needed to establish an active chromatin state in the RNA pol I gene promoterwhen the glucose concentration is raised after a period of deprivation. We propose that it counteracts the silent, poisedchromatin state imposed by the silencing chromatin remodelling complex NuRD to allow for the RNA pol I machineryto bind to the promoter.

These studies show that the influence of chromatin remodelling complexes upon gene expression is important forremodelling nucleosomes at the promoter, for alternative splicing, cleavage and polyadenylation and transcriptionalinitiation. These complexes work together with other chromatin remodelling factors, interact with other complexes andregulate their activity by affecting their recruitment dynamics.

Keywords: Chromatin remodelling, SWI/SNF, Brg1, Brm, alternative splicing, cleavage and polyadenylation, B-WICH,WSTF, ribosomal genes.

Stockholm 2018http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-161380

ISBN 978-91-7797-504-5ISBN 978-91-7797-505-2

Department of Molecular Biosciences, The Wenner-Gren Institute

Stockholm University, 106 91 Stockholm

GENE REGULATION BY CHROMATIN REMODELLINGCOMPLEXES

Antoni Gañez Zapater

Gene regulation by chromatinremodelling complexes

SWI/SNF complex in mRNA processing and B-WICH complex inribosomal gene expression

Antoni Gañez Zapater

©Antoni Gañez Zapater, Stockholm University 2018 ISBN print 978-91-7797-504-5ISBN PDF 978-91-7797-505-2 Printed in Sweden by Universitetsservice US-AB, Stockholm 2018 Cover and all figures in the introduction are from the author.

For all who helped me tofinish this

i

Sammanfattning

Målet med detta projekt är att förstå kromatinremodellerande komplex och

dess roll i genreglering. Två proteinkomplex står i fokus, BRG1 och BRM-

innehållande SWI/SN- komplex och det ISWI-innehållande B-WICH

komplexet.

Studie 1 undersöker SWI/SNF komplexens roll i alternativ splicing vid

mRNA transkription. De två ATPaserna BRG1 och BRM påverkar den

alternativa splicningen både så att fler exon inkluderas som att de

exkluderas. Då BRG1 och BRM båda är subenheter i

kromatinremodellerande komplex tror man att BRG1 och BRM påverkar

kromatin och transkriptionshastighet. BRG1 och BRM interagerar med en

rad proteiner, däribland proteiner som reglerar splicing och vi visar här att en

del av dessa interaktioner sker under transkription och leder till att olika

proteiner binder till RNA. Vi föreslår att detta skulle kunna vara en

mekanism som påverkar splicningen, och SWI/SNF komplexen kan på så

sätt vara med i att förändra isoformer i en liten grupp av proteiner. SWI/SNF

komplexen är avreglerade vid sjukdomar och detta kan leda till påverkan inte

bara på genexpression utan också på vilka isoformer som uttrycks.

Studie 2 undersöker rollen som SWI/SNF komplexens ATPaser spelar vid

terminering av mRNA-transkription. Vi har upptäckt att BRG1 och BRM

rekryterar klyvnings- och polyadenyleringsproteiner och därmed kan

termineringen öka av vissa transkript på speciella ställen. Detta har

betydelse för vilka isoformer som syntetiseras, men mer för stabilitet av

mRNA.

Studie 3 behandlar ribosomal transkription, vilken är tätt kopplad till cellens

metabolism och proliferation. Vi har upptäckt ett proteinkomplex, B-WICH,

som möjliggör transkription av dessa gener, så att celler kan producera

ii

ribosomer för proteinsyntes. Detta proteinkomplex remodellerar kromatinet

över promotorn som svar på höjd glukosnivå, vilket leder till att

transkriptionsmaskineriet kan binda in och börja transkribera. Ett annat

proteinkomplex, NuRD, sätter en kompakta struktur över promotorn när

transkriptionen skall gå ner, och B-WICH motverkar denna kompakta

struktur när aktiverande stimuli, som glukos, tillsättes.

Sammanfattningsvis bidrar dessa studier till en ökad förståelse av

kromatinremodellerande komplex och deras roll i olika processer, som

transkriptionsinitiering, splicing och polyadenylering av mRNA. SWI/SNF-

komplexen och B-WICH gör detta genom att remodellera kromatin men

också genom att interagera med andra komplex och protein.

iii

List of publications

Antoni Gañez Zapater, Sebastian Mackowiak, Yuan Guo, Antonio Jordán-

Pla, Marc Friedlander, Neus Visa, Ann-Kristin Östlund-Farrants. SWI/SNF

subunits Brg1 and Brm affect alternative splicing by changing RNA binding

factor interactions with RNA. Manuscript

Yu S, Jordán-Pla A, Gañez-Zapater A, Jain S, Rolicka A, Östlund Farrants

AK, Visa N. SWI/SNF interacts with cleavage and polyadenylation factors

and facilitates pre-mRNA 3' end processing. Nucleic Acids Res. 2018 May

31. doi:10.1093/nar/gky438. [Epub ahead of print] PubMed PMID:

29860334.

Anna Rolicka, Yuan Guo, Antoni Gañez Zapater, Jaclyn Quin, Anna

Vintermist, Fatemeh Sadeghifar, Marie Henriksson and Ann-Kristin Östlund

Farrants. The chromatin remodelling complex B-WICH is required for

ribosomal transcriptional activation by glucose stimulation. Submitted.

Work not included in this thesis

Eberle AB, Jordán-Pla A, Gañez-Zapater A, Hessle V, Silberberg G, von

Euler A, Silverstein RA, Visa N. An Interaction between RRP6 and

SU(VAR)3-9 Targets RRP6 to Heterochromatin and Contributes to

Heterochromatin Maintenance in Drosophila melanogaster.

PLoSGenet.2015Sep 21;11(9):e1005523. doi:10.1371/journal.pgen.1005523.

eCollection 2015 Sep. PubMed PMID: 26389589; PubMedCentral PMCID:

PMC4577213.

iv

Abbreviations

Brg1: Brahma-related gene 1 ATPase subunit of the SWI/SNF chromatin

remodelling complex.

Brm: Brahma ATPase subunit of the SWI/SNF chromatin remodelling

complex.

CPSF: Cleavage and Polyadenylation Specificity Factor.

CHD4: Chromodomain Helicase DNA Binding Protein 4.

DHX15: DEAH-box helicase 15.

GADD45A: Growth arrest and DNA-damage-inducible alpha.

HAT: Histone Acetyl Transferase.

HDAC: Histone Deacetylase.

hnRNPL: Heterogeneous nuclear ribonucleoprotein L.

hnRNPU: Heterogeneous nuclear ribonucleoprotein U.

ISWI: Imitation of Switch.

MAZ: Myc-associated Zinc Finger Protein.

mRNA: messenger RNA.

MYL6: Myosin light chain 6.

NuRD: Nucleosome Remodelling and Deacetylase complex.

PIC: Pre-initiation complex.

Pol I: RNA polymerase I.

Pol II: RNA polymerase II.

Pol III: RNA polymerase III.

rDNA: ribosomal RNA.

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RRN3: RNA polymerase I-specific transcription initiation factor RRN3.

Sam68: Src-associated Substrate in mitosis of 68 kDa.

SL1: Selectivity Factor 1.

SNF2h: Sucrose non-fermenting protein 2 homologue.

SWI/SNF: Switch/Sucrose non-fermentable chromatin remodelling complex.

SYF1: pre-mRNA splicing factor SYF1 (also known as XAB2).

TAF: TBP-associated factor.

TBP: TATA-binding protein.

UBF: Upstream binding factor.

WICH: WSTF-ISWI chromatin remodeling complex.

WSTF: Williams Syndrome Transcription Factor.

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Table of contents

Summary ................................................................................................... i

List of publications ................................................................................... iii

Abbreviations ............................................................................................ iv

1. Introduction ..................................................................1

Chromatin..................................................................................... 2

Chromatin remodelling ............................................................ 3

Histone modifications ............................................................. 3

ATP-dependent chromatin remodelling complexes ............... 5

SWI/SNF complex family .................................................... 6

Brg1 and Brm ATPase subunits ......................................... 8

ISWI-containing complexes family ...................................... 9

WSTF ................................................................................. 10

Protein coding genes ......................................................................... 11

RNA pol II transcription .............................................................. 11

mRNA processing ......................................................................... 13

Splicing .................................................................................... 13

The spliceosome and other splicing associated factors .......... 14

Alternative splicing ................................................................ 16

Cleavage and polyadenylation .................................................. 18

CPSF complex ........................................................................ 19

CstF, CFI, and CFII complexes .............................................. 19

SWI/SNF in RNA processing ....................................................... 19

Ribosomal genes ............................................................................... 21

RNA pol I transcription .................................................................. 22

RNA pol III transcription ................................................................ 23

B-WICH in regulation of ribosomal genes ..................................... 23

vii

2. Present investigation ....................................................25

Aim of the project ........................................................................... 25

Model system .................................................................................. 25

Study 1 ............................................................................................ 26

Study 2 ............................................................................................ 28

Study 3 ............................................................................................ 30

3. Conclusions ..................................................................32

4. Acknowledgements ......................................................33

5. References ..................................................................................... 34

1

1. Introduction

In higher eukaryotes, organism complexity does not depend only on the

number of genes but also on the way they are regulated. The genome is

packed in chromatin, and the level of this packing determines how factors

involved in gene expression are recruited to their target sites.

In general, chromatin can be classified depending on the level of

compaction: heterochromatin is a compacted conformation associated with

low levels of gene expression, and euchromatin is a more relaxed state that

allows genes to be accessible to factors that can alter gene expression

(reviewed in Huisinga et al. 2006). The level of compaction is regulated by

chromatin remodelling events, which can be classified into two major

groups: covalent histone modifications performed by histone modifying

enzymes, such as acetyltransferases (HATs) and methyltransferases (HMTs),

or by ATP-dependent chromatin remodelling complexes such as the

Switch/sucrose non-fermentable (SWI/SNF) complex or B-WICH (reviewed

in Voss and Hager 2014).

Chromatin remodelling can modulate the accessibility of chromatin to

multiple regulatory factors, therefore affecting both gene transcription and

later events in gene expression (reviewed in Braunschweig et al. 2013).

Transcripts coding for proteins also need to be processed by three major

mechanisms: 5’capping, splicing and polyadenylation (reviewed in Bentley

2014). These processes, particularly splicing and polyadenylation, can

introduce transcript variation. Through alternative splicing, exons can be

included or skipped from the final mRNA. In alternative polyadenylation,

different sites can be utilized for adding the polyadenylic acid (poly(A)) tail

at the 3’ end of the pre-mRNA, which can affect not only the coding region

but also the export and stability of the mRNA (reviewed in de Klerk and

Hoen 2015). In the first part of this project we investigate how the ATP-

2

dependent chromatin remodelling complex SWI/SNF influences processing

of transcripts to produce mature mRNA, taking into account both chromatin

features and interactions with other proteins.

It is essential that chromatin allows gene promoters to accessible to different

transcription factors when necessary, for example, to respond to changes in

conditions such as stress or growth signals. Ribosomes are essential for

synthesizing all the proteins in the cell. Mature mRNA associates with the

ribosome in order for it to be translated into its corresponding protein

(reviewed in Hershey et al. 2012). The genes coding for ribosomal RNAs

(rRNAs) exist in multiple copies (reviewed in Schöfer and Weipoltshammer

2018), and must be tightly regulated to rapidly respond to growth or stress

signals (reviewed in Gobet and Naef 2017). In the second part of this project,

we gain insights on how the chromatin remodelling complex B-WICH

influences the binding of other factors to the ribosomal genes to regulate

their expression.

Chromatin

Chromatin is a macro-complex comprised of proteins, DNA and RNA. Its

main function is to compact and protect genomic DNA through several

levels of condensation, which in turns becomes a regulation mechanism of

gene expression (reviewed in Venkatesh and Workman 2015).

The basic structure of chromatin is the nucleosome. Nucleosomes are

composed of eight histone proteins (two of each of H1, H2, H3 and H4),

which form a core histone that is wrapped by DNA. This first level of

chromatin compaction is 10nm wide and can be further compacted into a

30nm fiber composed of folded arrays of nucleosomes. The highest level of

compaction is usually achieved during mitosis and meiosis, when the 30nm

3

fibers are further folded until the mitotic chromosomes are formed (reviewed

in Luger et al. 2012).

Chromatin is usually divided in two major groups: a chromatin that is in an

open and relaxed state termed euchromatin, and a compacted and less

accessible state known as heterochromatin. Chromatin at active genes needs

to be in a de-compacted euchromatic state in order to make the DNA

accessible for the required factors. In general, euchromatin is associated with

transcriptionally active regions while heterochromatin is associated with

silenced regions. Nevertheless, this is not just an “ON/OFF” switch system

and other parameters, such as the spatial or temporal context, can affect

chromatin structure and the final levels of gene expression (reviewed in

Huisinga et al. 2006, Luger et al. 2012).

Chromatin remodelling

The structure of chromatin can be dynamically changed by chromatin

remodelling events in order to modulate gene expression. The two major

mechanisms for remodelling chromatin are by histone modifications and the

activity of ATPase dependent chromatin remodelling complexes, and both

mechanisms can work in a coordinated manner (Lavigne et al. 2009,

reviewed in Voss and Hager 2014).

Histone modifications

Histones can be modified by the addition or removal of different chemical

modifications, most frequently on the N-terminal histone tails. These

modifications affect the affinity of the histones with DNA or can work as a

signal for different chromatin factors. The final consequence is a change in

the chromatin structure (reviewed in Rothbart and Strahl 2014).

4

There is an increasing list of modifications that can take place at different

positions of the histone tails, but the most extensively studied are

methylation, acetylation, phosphorylation and ubiquitination. Some

examples of well characterized modifications that are important in gene

expression are H3K4me3, H3K9ac and H3K14ac in actively transcribed

promoters, H3K36me3 in actively transcribed gene bodies, H3K9me3

associated with heterochromatin, and H3K27me in facultative repressed

genes (reviewed in Böhm and Östlund-Farrants 2011, reviewed by Zhao and

Garcia 2015).

Figure 1. Different chromatin states regulated by histone modification and

chromatin remodelling complexes (reviewed in Layman and Zuo 2015).

5

Histone modifications are added or removed by specific enzymes. The most

extensively studied groups of these enzymes are the histone

methyltransferases (HMTs), acetyltransferases (HATs), deacetylases

(HDACs) and demethylases (HDM) (reviewed in Marmorstein and Trievel

2009). The regulation of these enzymes is an important layer of gene

expression regulation.

ATP-dependent chromatin remodelling complexes

The ATP-dependent chromatin remodelling complexes use the energy from

the hydrolysis of ATP in order to alter DNA-histone contacts. There are at

least four families in which the chromatin remodelling complexes can be

grouped: SWI/SNF, ISWI, CHD and INO80. Although they share basic

properties including affinity for DNA, affinity for nucleosomes, capacity for

recognizing histone modifications, and an ATPase catalytic domain, they

differ in other subunits and domains. These variations make possible

multiple ways of regulating the ATPase as well as by interacting with other

factors and allowing the crosstalk between major cellular processes

(reviewed in Mayes et al. 2014, Clapier and Cairns 2009).

Figure 2. Composition of the four main families of chromatin remodelling

complexes and characteristic domains present (adapted from Mayes et al

2014).

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SWI/SNF complex family

The SWI/SNF complexes are composed of core and accessory subunits. In

humans, the core is typically formed by one of the two ATPase catalytic

subunits Brg1 (SMARCA4) or Brm (SMARCA2), BAF155 (SMARCC1),

BAF170 (SMARCC2) and Ini1/SNF5 (SMARCB1). In human, there are two

predominant subfamilies of SWI/SNF complexes: BAF (Brg1/hBrm-

Associated Factors) and PBAF (Polybromo-associated BAF). The main

differences between these subfamilies are that BAF complexes can have

Brg1 or Brm as ATPase subunits while PBAF only contains Brg1, and that

BAF250 can only be found in BAF while Polybromo/BAF180 can only be

found in PBAF. Other constellations have also been observed to exist, such

as sub-complexes containing subunits from both subfamilies (Ryme et al.

Figure 3. Scheme of the composition of the two main families of

mammalian SWI/SNF complexes, BAF and PBAF. The characteristic

subunits of each family are shown in white, ATPase subunits BRG1 and

BRM in black, core subunits in dark grey and other accessory subunits in

light grey (adapted from Hohmann and Vakoc 2014).

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2009). Other subfamilies can be associated with other factors, i.e. BRCA1,

and some, like enBAF, are tissue specific (reviewed in Mohrmann and

Verrijer 2005, Hohmann and Vakoc 2014, Kadoch and Crabtree 2015).

SWI/SNF complexes are distinguished from the other ATPase complexes by

having ATPase subunits that have a bromodomain. The bromodomain

recognizes acetylated lysines (Dhalluin et al. 1999). It is also present in

many HATs and other chromatin binding proteins like Polybromo, where

several copies are present, suggesting that this subunit targets PBAF to open

acetylated chromatin regions (reviewed in Mohrmann and Verrijer 2005).

Nevertheless, BAF complexes with mutant Brm lacking a bromodomain can

also bind to acetylated chromatin regions, suggesting that other factors

collaborate in this binding (Hassan et al. 2002). ARID (A-T rich interaction

domain) is another relevant DNA-binding domain present in BAF250 that, in

this protein, works in a sequence-unspecific manner. BAF250 has been

implicated in the transcriptional activation of hormone receptors, but this

specificity is not given by the ARID domain, suggesting that it binds to other

cofactors (reviewed in Mohrmann and Verrijer 2005). SWI/SNF does not

work as a single entity, but rather as a toolbox with specialized features in

each subunit. This also opens the possibility that SWI/SNF subunits can

work independently of the SWI/SNF complex environment and have roles in

other processes (Euskirchen et al. 2011, reviewed in Savas and Skardasi

2018).

SWI/SNF complexes have important roles in differentiation and proliferation

(reviewed by Ko et al. 2008, reviewed in Masliah-Planchon et al. 2015).

Deficiencies in the SWI/SNF subunits can impair embryonic development,

induces peri-implantation lethality and also can arrest the proper

development of T-cells (reviewed by Ko et al. 2008, Mohrmann and

Verrijzer 2005). SWI/SNF can be essential for cell survival but can also

induce senescence, and this dual role is most probably due to binding to

8

different factors present in each context (reviewed in Mohrmann and

Verrijer 2005). This role in modulating cell proliferation or growth arrest has

been widely studied for its implications in cancer. SWI/SNF members are

found to be mutated or their function is affected in many cancers (reviewed

in Ko et al. 2008, Kadoch and Crabtree 2015, Wilson and Roberts 2011),

therefore SWI/SNF in general is considered a tumour suppressor. Some of

the proteins implicated in tumorigenesis found to be associated with

SWI/SNF are Retinoblastoma protein (pRB), Breast cancer type 1 (BRCA1),

Lysine methyltransferase 2A (KMT2A, also known as MLL) and h-catenin

(reviewed in Roberts and Orkin 2004). Gene expression might not be the

only role of SWI/SNF in developing cancer. In yeast, the RSC complex,

highly similar to SWI/SNF, is involved in the segregation of sister

chromatids during mitosis, and recent studies in human cells showed PBAF

in kinetochores, opening the possibility that SWI/SNF might be involved in

cell proliferation not just by regulating gene expression but also mitosis (Xue

et al. 2000, Euskirchen et al. 2011).

Brg1 and Brm ATPase subunits

Brg1 and Brm, the ATPase subunits of mammalian SWI/SNF, are

biochemically very similar proteins. As the ATPase subunits from the other

chromatin remodelling complexes, Brg1 and Brm catalytic site is composed

by a DExx and a Helicase domain spaced by a linker. The characteristic

domains for Brg1 and Brm are the QLQ domain, needed for binding with

other proteins, the HAS domain for to b-actin and BAF53a, the BRK domain

of unknown function, and the bromodomain which binds to acetylated

histones (reviewed in Tang et al. 2010). Brg1 and Brm only differ in the fact

that Brg1 has a unique N-terminal domain that binds to Zn fingers, which is

thought to allow the interaction with different proteins (Kadam and Emerson

2003).

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The in vivo implications are different (reviewed in Hohmann and Vakoc

2014). Brg1 knock-out mice die during early embryogenesis (Bultman et al.

2000). Initial studies reporting lack of this phenotype in Brm knock-out mice

need to be revised due to discovery of functional truncated Brm protein in

this model (Thompson et al. 2015). Brg1 and Brm induce different kinds of

cancer when mutated or lost (Masliah-Planchon et al. 2014, reviewed in

Mohrmann and Verrijer 2005, reviewed in Savas and Skardasi 2018), are

targeted to double strand brakes and are involved in the DNA repair (Qi et

al. 2015, Kwon et al. 2015), and they also play a role in the organization of

the actin cytoskeleton (Asp et al. 2002).

ISWI-containing complexes family

ISWI (imitation of switch) is a SWI2/SNF2 ATPase that is homologous to

Brg1 and Brm. Two ISWI have been identified in human: SNF2h (imitation

of switch homolog) and SNF2l (imitation of switch like). These ATPase can

be found in a family of complexes characterized for having two or three

subunits and a large subunit with histone-binding domains including PHD

and bromodomains (reviewed in Mayes et al. 2014). The ISWI chromatin

remodelling complexes slide nucleosomes in short increments and there are

specialized complexes involved in different processes: CHRAC and ACF are

involved in spacing nucleosomes after replication; RSF in RNA polymerase

elongation; CERF, NURF, NoRC, WICH and B-WICH are co-regulators of

transcription and WICH is also involved in DNA damage repair (reviewed in

Mayes et al. 2014).

Here we focus on B-WICH, an extended form of WICH (composed of the

core subunits WSTF and SNF2h), that additionally contains Nuclear Myosin

1 (NM1) (Percipalle et al. 2006, Cavellán et al. 2006, reviewed in Barnett

and Krebs 2010). B-WICH is known to regulate RNA polymerase III during

the transcription of the non-coding RNAs 5S and 7SL (Cavellán et al. 2006)

10

by maintaining an open chromatin state that allow the binding of other

activating factors like c-Myc (Sadeghifar et al. 2015). B-WICH also

regulates RNA polymerase I transcription of 45S RNA (Percipalle et al.

2006), induces changes in the chromatin structure of ribosomal genes and

allows for histone acetyl transferases (HATs) to bind and maintain an active

chromatin state (Vintermist et al. 2011).

WSTF

WSTF (the BAZ1B gene) is a subunit of the WICH and B-WICH

complexes. WSTF has tyrosine kinase activity that regulates the H2A.X

DNA damage response (Xiao et al. 2009), is involved in the regulation of

transcription of small non-coding RNA by RNA polymerase III (Cavellán et

al. 2006) and regulates transcription of ribosomal genes by RNA polymerase

I (Percipalle et al. 2006, Vintermist et al. 2011). WSTF has several

conserved motifs (reviewed in Barnett and Krebs 2011). The WAC domain

has tyrosine kinase activity (Xiao et al. 2009) and can bind to DNA

(Fyodorov and Kadonaga 2002). The PHD domain has a zinc-binding motif

that can read the methylation state of H3K4 and is related to gene activation

depending on the context (reviewed in Sanchez and Zhou 2011). The

bromodomain is an acetyl-lysine reader and binds WSTF to chromatin

(Dhalluin et al. 1999).

WSTF was discovered as being deleted in patients with Williams syndrome,

who have a deletion of approximately 1.5 Mb in chromosome 7 that has

about 28 other genes (Lu et al. 1998, reviewed in Schubert 2009). These

patients suffer from cardiovascular disease, intellectual disability and

endocrine abnormalities among others (reviewed by Morris 1999), however,

it is difficult to assign how many of these characteristics are due to lack of

only WSTF.

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Protein coding genes

Protein coding genes are transcribed into messenger RNA (mRNA) by the

RNA polymerase II, and this mRNA is then translated into a protein by a

ribosome in the cytosol. The region of the mRNA that precedes the start

codon (“upstream”) and is not translated is termed 5’ untranslated region

(5’UTR) and the one that proceeds the stop codon (“downstream”) is the 3’

untranslated region (3’UTR). In between start and stop codons there are

introns and exons. The introns are removed from the pre-mRNA before

translation and therefore are not reflected in the protein. The 5’UTR, 3’UTR

and introns, although not included in the protein sequence, have important

regulatory roles in the maturation of the mRNA (reviewed in Müller-

McNicoll and Neugebauer 2013).

Polymerase II transcription

RNA Polymerase II (Pol II) transcribes pre-mRNA from protein coding

genes and the majority of small nuclear RNA (snRNA) and microRNA

genes (reviewed in Guiro and Murphy 2017).

The transcription machinery is composed of the Pol II and additional

transcription factors that differ depending if the transcription is in the

initiation, elongation or termination stage (reviewed by Hanh 2004).

RNA Pol II is composed of 12 subunits. It cannot bind to the promoter

sequences by itself, but needs the recruitment of general transcription factors

(reviewed in Thomas and Chiang 2006). Together with TFIIA (Transcription

Factor II A), TFIIB, TFIID, TFIIE, TFIIH, and Mediator, Pol II forms the

pre-initiation complex (PIC) (reviewed by Hanh 2004). These accessory

transcription factors help Pol II to position correctly at the promoter by

recognizing certain sequences. Some of these sequences include the TATA

12

element (recognized by TBP, a subunit of TFIID), the BRE (TFIIB

recognition element), the Inr (Initiator) and the DPE (downstream core

promoter element) (reviewed by Hanh 2004, reviewed in Thomas and

Chiang 2006). These elements can be found in different combinations in the

promoter and none of them are essential (reviewed in Smale and Kadonaga

2003).

An important element for the regulation of the Pol II is the C-terminal

domain (CTD). The CTD is a sequence of amino acids, YSPTSPS, repeated

around 52 times in human Pol II (Broyles and Moss 1986). During initiation,

the CTD Ser5 is phosphorylated to keep a paused state, and is gradually

dephosphorylated during elongation. On the other hand, the Ser2 is

phosphorylated by the kinase P-TEFb during transcription to maintain the

elongation state (reviewed in Harlen and Churchman 2017, reviewed in

Chen et al. 2018). The CTD also works as scaffold for other factors that are

needed for 5’ capping, splicing, 3’ end processing and termination (reviewed

in Meinhart et al. 2005).

During transcription elongation, the RNA molecule is synthesized but the

rate can be affected due to the chromatin landscape as well as the

phosphorylation state of the Pol II (reviewed in Chen et al. 2018). This can

have consequences in downstream processes such as splicing, where the

elongation rate can influence alternative exon inclusion (reviewed in

Naftelberg et al. 2015).

Less is known about the termination of transcription of Pol II in eukaryotes.

The model for coding genes is termed poly(A)-dependent, or torpedo model,

which involves cleavage of the mRNA between the poly(A) signal and GU-

rich region followed by degradation of the RNA still bound to the Pol II by

XRN2 (5’-3’ exoribonuclease 2). For non-coding genes in yeast the Sen1-

dependent model has been proposed, consisting in recruitment of Sen1

13

proteins bound to CTD and specific RNA sequences and unwinding of the

Pol II by the helicase activity of Sen1 (reviewed in Kuehner et al. 2011).

mRNA processing

The pre-mRNA is the newly transcribed protein coding RNA, and must be

modified by several mechanisms before becoming mature mRNA that is

translated into protein in the cytoplasm. The three major steps in this

processing are the 5’ capping, splicing and polyadenylation (reviewed in

Müller-McNicoll and Neugebauer 2013). In higher eukaryotes, the

regulation of splicing and polyadenylation are key points that can be

regulated in alternative ways, giving a variable number of different

transcripts with different biological consequences (reviewed in Müller-

McNicoll and Neugebauer 2013). The regulation of alternative splicing and

polyadenylation is influenced by other pathways, such as chromatin

remodelling (Luco et al. 2010, reviewed by Braunschweig et al., 2013,

(reviewed in Müller-McNicoll and Neugebauer 2013), to achieve multiple

levels of regulation of gene expression in a context specific manner.

Splicing

Splicing involves two transesterification steps. The mRNA is first cleaved at

the 5’ splice site (5’ss) of the intron and is ligated to the branch site (BS),

which is typically located 18-40bp upstream the 3’ splice site (3’ss), forming

a loop. Second, the intron is removed from the mRNA by cleaving in the

3’ss and the two harbouring exons are ligated (reviewed in Will and

Lührmann 2006).

14

The spliceosome and other splicing associated factors

The spliceosome is the complex in charge of performing splicing. It is a

dynamic complex of snRNA and proteins that form the small nuclear

ribonucleoprotein (snRNP). Other factors, such as members from the hnRNP

(heterogeneous nuclear ribonucleoprotein) and SR (serine/arginine) protein

families, bind at specific stages of the splicing process (Hegele et al. 2012).

The assembly of all the elements is a step-wise process where the different

snRNA and splicing factors are recruited and released depending on the

stage of the splicing process, forming several intermediate complexes

(reviewed in Will and Lührmann 2011).

The snRNA are small RNA molecules of approximately 150 nucleotides that

are located in nucleus and enriched in sub-nuclear structures, the speckles

and Cajal bodies in eukaryotic cells (Machyna et al., 2013).

Figure 4. Steps of splicing process showing dynamic incorporation and

release of the splicing factors (adapted from Will and Lührmann 2011).

15

The snRNA characterize two major kinds of spliceosomes: the U2 and the

U12-dependent. The U2-dependent spliceosome, the predominant form, is

composed of the U1, U2, U4, U5 and U6 snRNA, and is in charge of

splicing the U2-type introns, characterized by a GT dimer at the 5’ss and a

AG at the 3’ss. U12-dependent spliceosome, or “minor spliceosome”, which

also exists in metazoans, is formed by the U11, U12, atacU4 and atacU6

snRNAs and performs the splicing of a less abundant U12-type introns

characterized by a AT and AC dimers at the 5’ss and 3’ss respectively

(reviewed by Patel and Steitz 2003).

The snRNAs bind to two families of proteins, the Sm and LSm (like-Sm),

and form a structure conserved from yeast to humans. The assembly of these

snRNP in a proper structure is essential for the coordination with other

subcomplexes involved in the spliceosome assembly (reviewed in Will and

Lührmann 2001).

The hnRNP family includes proteins that have the function of binding to

exonic splicing silencer (ESS) cis-elements, most commonly through the

RNA recognition motif (RRM). There is not a single mechanism by which

the hnRNP inhibit splicing. Different strategies that have been described

include the repression of spliceosomal assembly, blocking the recruitment of

snRNP, or looping out exons (reviewed in Busch and Hertel 2012).

The SR family includes factors involved in the recruitment of the splicing

machinery to the splice sites. They are characterized by an arginine/serine

rich (RS) domain and a RRM domain. SR proteins recognize exonic splicing

enhancers (ESE), cis-elements in the mRNA that promote exon inclusion

through their RRM domain. Then SR proteins can induce inclusion of the

alternative exon by recruiting splicing factors to the weaker splice site,

through their RS domain (reviewed in Black 2003, Busch and Hertel 2012).

16

As previously mentioned, the splicing process involves a large amount of

proteins that are recruited and released during the different steps (Hegele et

al. 2012). 200 proteins that co-purify with the splicing machinery have been

identified so far, but not all are included in the hnRNP and SR families

(reviewed in Wahl et al. 2009). For instance, the pre-mRNA-processing

factor 19 (PRP19) complex, which is involved in the assembly and the

activity of the spliceosome and has been suggested to have a role in coupling

transcription and splicing (David et al. 2011). The DEAD-box helicase

(DDX), which contains numerous helicases that separate duplex

oligonucleotides and have important roles in RNA metabolism (reviewed in

Gustafson and Wessel 2010). The exon junction complex (EJC) and the

transcription and export complex (TREX) which work closely together in

order to prevent premature export of non-spliced RNA (reviewed in Pawlicki

and Steitz 2010).

Alternative splicing

About 95% of multi-exon genes in humans undergo alternative splicing

(Wang et al. 2008). It is a tightly regulated mechanism that provides a

significant source of variability and adaptability by producing different

transcript isoforms from a single gene, with putative different functions

(Nilsen and Graveley 2010).

Alternative splicing events can be classified into different groups: the most

common are exon skipping, mutually exclusive exons, and intron retention,

while alternative 5’ donor site and alternative 3’ acceptor site are less

common. Furthermore, more than one type of alternative splicing event can

be found in the same gene (reviewed in Blencowe 2006). Each depends on

the exon and intron definition by both regulation of the splicing and other

accessory factors, and cis-regulatory elements in the sequence that can be

recognized by the SR and hnRNP families (reviewed in Busch and Hertel

17

2012). The context, including the chromatin structure and epigenetic marks,

can also have indirect roles.

There are two models explaining alternative splicing: the recruitment model

and the kinetic model (reviewed in Kornblihtt 2007, Braunschweig et al.

2013, Naftelberg et al. 2015). In the recruitment model, the activity of

splicing factors at the splicing site is mediated by recruitment to the site by

other factors. This step-wise binding can involve many different proteins,

having many different roles in the process. In the kinetic model, the activity

of splicing factors at a splice site depends upon how long the splicing factors

bind to the splice site. For example, weak splice site might need more time

to be recognized by splicing factors; therefore, if the transcription and

splicing are taking place in a fast manner, only strong splice site would be

recognized; while on the other hand, if the weak splice sites are accessible

for sufficient time to be recognized, they might be used as well (reviewed by

Kornblihtt 2007).

Figure 5. Different transcripts resulting from alternative splicing events

(adapted from Cartegni et al 2002).

18

Cleavage and polyadenylation

A poly(A) tail is required to be added at the 3’ of almost all eukaryotic

mRNA during mRNA processing. This is an important signal for nuclear

export, translation and stability of the mRNA (reviewed in Müller-McNicoll

and Neugebauer 2013).

The pre-mRNA is cleaved in the cleavage site and then the poly(A) tail is

synthesized at the 3’ end. These two steps are regulated by cis-elements in

the pre-mRNA and several trans-acting factors. The complexes that form the

core machinery are the cleavage and polyadenylation specificity factor

(CPSF), the cleavage stimulation factor (CstF), and the cleavage factors I

and II (CFI and CFII). Other protein factors involved are the poly(A)

polymerase (PAP), Symplekin and the C-terminal domain of the largest

subunit of the Pol II (CTD) (reviewed in Mandel et al. 2008). Nevertheless,

recent studies identified new factors related to the 3’ processing machinery

like PP1, WDR33 or RBBP6, as well as factors related to other processes as

splicing, RNA quality control or DNA damage that can mediate this process

(reviewed in Braunschweig 2013, Di Giammartino and Manley 2014).

Figure 6. Factors involved in cleavage and polyadenylation events

(adapted from Elkon et al 2013).

19

CPSF complex

The CPSF complex recognizes the AAUAAA hexamer in mammals, known

as the polyadenylation signal (PAS). The CPSF complex is composed of

CPSF1, CPSF2, CPSF3, CPSF4, FIP1 and WDR33. CPSF1 is the largest

subunit and in charge of recognizing the PAS, although it alone cannot

explain all the affinity of the complex for this sequence. Recent experiments

show that WDR33 and CPSF4 also can bind PAS in vitro (Chan et al. 2014;

Schonemann et al. 2014), and that Fip1 recognizes sequences upstream PAS

(Chan et al. 2014), suggesting that they also have a role in the recognition of

PAS in vivo (reviewed in Shi and Manley 2015). The cleavage is performed

by CPSF3 at the cleavage site, which is usually a CA dinucleotide at 10 to

30 nucleotides from the PAS.

CstF, CFI, and CFII complexes

CstF, through CstF64 or CstF64τ, recognizes the downstream element

(DSE), a G/U rich region at less than 30 nucleotides downstream of the

cleavage site. The CFI complex contains the CPSF5, CPSF6 and CPSF7, and

recognizes UGUA motifs upstream of the PAS (reviewed in Shi and Manley

2015). The CFII seems to associate with the other complexes transiently,

since it is not clearly detected in proteomic analysis of purification of the 3’

processing complex (Shi et al. 2009), and its function might be the

connection to other pathways (reviewed in Di Giammartino and Manley

2014).

SWI/SNF complexes in RNA processing

Splicing and polyadenylation are mainly co-transcriptional events, and this

means that these can be regulated by the different mechanisms involved in

20

transcription and chromatin (Luco et al. 2010, reviewed in Braunschweig et

al. 2013).

It is known that a downstream exon can be alternatively spliced depending

on how a promoter drives transcription (Cramer et al. 1997). One of the main

functions of the ATPase chromatin remodelling complex SWI/SNF is

regulation of promoter accessibility. Furthermore, it has been shown that

SWI/SNF components extensively co-localizes with RNA polymerases II

and III (Euskirchen et al. 2011) and that it binds to nascent mRNA co-

transcriptionally (Tyagi et al. 2009).

It has been suggested that SWI/SNF ATPases can affect alternative splicing

by binding to known splicing factors (Ito et al. 2008) and to Pol II in an

elongation-dependent manner (Batsché et al. 2006). The ATPase catalytic

activity of Brm is not necessary to modulate the alternative splicing of the

Drosophila CD44 gene by this subunit (Batsché et al. 2006). Nevertheless,

deleting the Brm bromodomain, which necessary for the binding of Brm to

acetylated histones, has an inhibitory effect on splicing probably due to the

sequestration of splicing factors and the inability of SWI/SNF subunits to

bind to the promoter regions. An accumulation of Pol II Ser5

phosphorylation, which is associated with paused polymerase, was also

observed in the variant exons affected by Brm, suggesting that SWI/SNF has

a role in the phosphorylation of the Pol II (Batsché et al. 2006).

In the model proposed, the slowdown of polymerase elongation combined

with the recruitment of splicing factors by SWI/SNF complexes may explain

the effect of Brm in the different exon inclusion of the CD44 gene (Batsché

et al. 2006). Drosophila studies suggest that this is not a general mechanism

of splicing regulation, but only occurs at certain loci, and other subunits of

the SWI/SNF, such as SNR1, are also essential in this process (Waldholm et

al. 2011; Zraly and Dingwall 2012, Yu et al. 2014).

21

Less is known about the role of SWI/SNF complexes in polyadenylation, but

there are also reported connections. For instance, a model is proposed in

yeast where the Sin1p chromatin protein and the SWI/SNF complex, among

other protein factors, are needed for the recruitment of the cleavage and

polyadenylation complex (Hershkovits et al. 2006). Furthermore, some

indirect effects on polyadenylation could be actually taking place via the

connection to other pathways, for example splicing. Splicing and

polyadenylation are known to compete for cis-regulatory elements (Tian et

al. 2007, reviewed in Braunschweig et al. 2013) and several proteins are

involved in both processes (Shi et al. 2009, Huang et al. 2012).

Ribosomal genes

Ribosomes are in charge of translation, which is the process of the codon

sequence of an mRNA being made into the corresponding chain of amino

acids, to produce the encoded protein. Due to the need for ribosomes to

make new proteins, they are essential for the cell. Ribosomes are composed

of RNA (rRNA) and proteins. Ribosomal rRNA need to be produced in high

amounts representing up to 70% of all cell transcription. The ribosomal

genes (rDNA) are transcribed by Pol I and Pol III, to produce the 45S and 5S

rRNA respectively. 45S pre-rRNA is transcribed from up to 400 copies (not

all active simultaneously) of 43kb rDNA genes (rDNA) located in tandem

repeats in chromosomes 13, 14, 15, 21 and 22, that form the nucleolar

organizer regions (NORs) (Sanij et al. 2008, reviewed by Birch and

Zomerdijk 2008, reviewed in Goodfellow and Zomerdijk 2013) . The 45S is

processed into the 5.8S, 18S and 28S rRNA. The 5S is transcribed from

2.2kb gene locus containing around 3000 copies placed in tandem repeats on

chromosome 1, a region that does not form a NOR (Stults et al. 2008). The

5S rRNA and ribosomal proteins have to be exported to the nucleolus,

formed around the NOR, where together with the mature 5.8S, 18S and 28S

22

rRNA formed from the 45S pre-rRNA, they assemble into ribosomes

(reviewed by Moore and Steitz 2002).

Polymerase I transcription

The RNA polymerase I (Pol I) is in charge of transcribing the 45S rRNA.

Similarly to other RNA polymerases, the Pol I needs to form a pre-initiation

complex (PIC) before starting the transcription. The PIC is composed of Pol

I as well as UBF (Upstream binding factor), SL1 (Selectivity factor 1,

composed of TATA binding protein TBP and four TBP associated factors)

and RRN3 (also known as TIF1A) (reviewed in Goodfellow and Zomerdijk

2013). UBF and SL1 co-stabilize each other’s binding at the promoter, and

recruit Pol I associated with RRN3, which is able to initiate transcription.

RRN3 is released to allow the Pol I to transcribe, and this is achieved by the

phosphorylation of two specific serines by CK2 (Bierhoff et al. 2008). UBF

also binds across the transcribed region of the rDNA to maintain its

euchromatic state. Regulation of Pol I transcription can occur at multiple

steps, including PIC assembly, initiation and promoter escape, elongation,

and transcription termination (reviewed in Goodfellow and Zomerdijk 2013).

Multiple factors contribute to facilitate transcription, which can directly

target Pol I and its transcription factors, including SL1, UBF, RRN3, as well

as chromatin remodelling factors that mediate the transcription between

poised and active state at the rDNA promoter, including CSB (Cockayne

syndrome protein B) which associates with G9a histone methyltransferase

and B-WICH (Yuan et al. 2007, Cavellán et al. 2006).

The transcription by Pol I terminates when TTF-1 (transcription termination

factor) recognizes the Sal box in mammals, and the rRNA is released when

PTRF (Pol I and transcript release factor) associates with Pol I and TTF-1

(reviewed in Richard and Manley 2009).

23

Polymerase III transcription

The RNA polymerase III (Pol III) transcribes short non coding RNAs,

including the 5S rRNA, the tRNA, U6 RNA or the 7SL genes. It is the

largest of the three RNA polymerases but synthesizes RNA of only around

100 - 150bp. On the other hand, it is rapidly reassembled and can produce

huge amounts of these RNA (reviewed in Richard and Manley 2009).

In mammals, genes transcribed by Pol III can be classified in three types

depending on the features of their promoters. Type 1 is the promoter of 5S,

which is downstream the transcription start site. It contains an A box, C box

and the intermediate element (IE) that are recognized sequentially by TFIIIA

(exclusive for type 1 promoters), TFIIIC and TFIIIB containing TBPs. Type

2 promoters are present in tRNA genes, and similarly to type 1 have internal

A and B boxes, but these are only recognized by TFIIIC, which then recruits

TFIIIB. Type 3 promoters have external regulatory sequences consisting in a

TATA box, a proximal sequence element (PSE) and a distal sequence

element (DSE). The Pol III is recruited to the promoter and transcription can

start when the TBP is bound to the TATA box and the PSE-binding protein

(PBP) or the snRNA-activating complex (SNAPc) are in the PSE element

(reviewed in Dumay-Odelot et al. 2010).

Pol III transcription terminates when it comes upon a cluster of T. Pol III can

terminate transcription on its own, however, there are factors like

Topoisomerase I, nuclear factor 1 (NF1) and La protein that are suggested to

facilitate the release of the 5S RNA from the Pol III (reviewed in Richard

and Manley 2009).

B-WICH in the regulation of ribosomal genes

Ribosomal genes are present in multiple copies in human cells. The

approximately 400 copies of the 45S rDNA and 300 copies of 5S (Stults et

24

al. 2008) are not active at the same time, and their expression need to be

tightly regulated.

B-WICH is known to bind to Pol I, is present at both promoter and the

coding region of the 45S rDNA, and facilitates Pol I transcription on the

chromatin (Percipalle et al. 2006, Cavellán et al. 2006). B-WICH regulates

the transcription from 45S rDNA by remodelling a specific region of 200bp

around the promoter, which facilitates the binding of UBF to the UCE

sequence. The B-WICH subunit WSTF promotes an increase in the levels of

H3K9 acetylation by facilitating the access to specific H3 acetyl-

transferases, which results in the activation of the ribosomal genes

(Vintermist et al. 2011). This permissive state is ensured by the association

of NM1 to the rDNA, which also cooperates with b-actin to preserve an

epigenetic landscape compatible with Pol I transcription (Almuzzaini et al.

2016).

B-WICH is also present in genes transcribed by Pol III, including the 5S

rDNA, and regulates their transcription through interactions with other

nuclear proteins (Cavellán et al. 2006). B-WICH changes the chromatin

structure close to the 5S rDNA during Pol III transcription and is required

for the binding of the Pol III machinery. A model proposed that B-WICH

facilitates the recruitment of the c-Myc-Max complex to the IGS, which in

turn recruits HATs to the 5S rDNA and therefore maintaining an active state

along the gene (Sadeghifar et al. 2015).

25

2. Present investigation

Aim of the project

The aim of this project is to investigate the roles of chromatin remodelling

complexes in gene regulation. In the first part, we analysed the roles of

SWI/SNF ATPases in two key processes of mRNA maturation: cleavage and

polyadenylation of the 3’end, and splicing. In the second part, we studied the

role of B-WICH on the regulation of ribosomal genes upon glucose

stimulation.

Model system

These studies were carried out in tissue culture cells from man and

Drosphila. Human cancer cervix Hela cell line was used in all the studies.

C33A is another cervix cancer cell line which is deficient in the expression

of the SWI/SNF subunits Brg1 and Brm and was used in studies 1 and 2.

Drosophila S2 cells are derived from a primary culture of late stage-embryo

and were used in Study 2.

26

Study 1

SWI/SNF subunits Brg1 and Brm affect alternative splicing by

changing RNA binding factor interactions with RNA.

Aim

SWI/SNF chromatin remodelling complexes are known for remodelling

nucleosomes at promoter region to allow for gene expression. It was also

found to regulate alternative splicing of subsets of genes by modulating the

Pol II transcription dynamics and by interacting with splicing associated

factors. The aim of Study I is to investigate the influence of SWI/SNF

catalytic subunits Brg1 and Brm in alternative splicing genome wide and to

study the roles of the interactions with known alternative splicing factors.

Results

In Study 1 we show that SWI/SNF catalytic subunits Brg1 and Brm affect

the alternative splicing of a subset of genes and interact with alternative

splicing factors in the nascent mRNA.

We started by expressing Brg1 and Brm, the catalytic subunits of SWI/SNF,

in the C33A cell lines, which lack of these two proteins. RNA-seq analysis

revealed a subset of genes to be affected, promoting inclusion of

alternatively spliced exons in the majority of the cases. We observed that

more included exons had a higher GC content and higher difference between

the GC content between the exon affected and harbouring introns, than

skipped exons. In contrast, using K562 publically available data, we

observed that skipped exons had higher nucleosome occupancy as suggested

in previous studies (Amit et al. 2012). We investigated the mechanism

behind the inclusion promoted by SWI/SNF by investigating three genes:

MYL6, MAZ and GADD45A. Expression of Brg1, Brm and their ATPase

27

mutated versions induced changes in the H3 density in the affected exons,

suggesting an induction to open chromatin. No changes were found,

however, in the Pol II occupancy, and no general change in the

phosphorylation levels of RNA pol II CTD Ser2 or Ser5 in these exons

occurred. These results indicate that the transcription dynamics were not

affected at the sites, which has been reported for BRM in previous studies in

human cells or Drosophila (Batschè et al. 2006, Zraly and Dingwall 2009).

To further investigate this discrepancy in RNA pol II occupancy and

phosphorylation level of the CTD, we analysed the interactions of Brg1 and

Brm with known splicing factors in the nascent RNA and analyzed their

roles in the alternative splicing of MYL6, MAZ and GADD45A. We found

that knock down of Sam68, hnRNPU, hnRNPL, DHX15 and SYF1 had

different roles affecting these exons. Finally, we found that Brg1 and Brm

can regulate the recruitment of these splicing factors to the target exon at

both chromatin and nascent RNA level. Interestingly, most of the factors

investigated was recruited to chromatin by Brg1 and Brm, but the binding to

nascent RNA was more discriminating and even different upon Brg1 and

Brm expression.

Our RNA-seq data identified novel SWI/SNF complex target sites and we

cannot exclude the possibility that SWI/SNF affects alternative splicing by a

slightly different mechanism in other contexts than the one found in our

model genes. We suggest that SWI/SNF has a more direct role in alternative

splicing than previously shown by regulating the recruitment of different

splicing factors to their target sites. We propose that Brg1 and Brm in the

context of a SWI/SNF complex at the exon alters the interactions between

RNA regulators and general splice factors, in particular those to the nascent

RNP. In that way, exon inclusion and skipping are promoted and the level of

alternative isoforms is fine-tuned.

28

Study 2

SWI/SNF interacts with cleavage and polyadenylation factors and

facilitates pre-mRNA 3' end processing.

Aim

Cleavage and polyadenylation are essential steps in the maturation of mRNA

that can have important regulatory consequences for the cell. In Study 2 we

aim to investigate the interaction of SWI/SNF with the CPSF complex and

how this affects with the cleavage and polyadenylation efficiency in a subset

of genes.

Results

In Study 2, we used the both Brg1 and Brm deficient cell line C33A and

Drosophila S2 cells to study the generality of mechanisms in the cleavage

process. We found that Drosophila Brm (dBrm) and human Brg1 (hBrg1)

facilitate the 3’end cleavage of specific genes and that SWI/SNF was present

in the target place. An RNA-seq performed after knocking down dBrm

revealed an increase of signal after the cleavage site in a subset of genes,

named Cluster 1, independently of the changes in general gene expression,

suggesting 3’end processing defects. ChIP-seq analysis showed that in

Cluster 1 genes dBrm was present downstream of the cleavage site, and

publically available data showed a lower nucleosome density than in other

clusters, suggesting that in these genes dBrm is needed to maintain an open

chromatin state. We also identified dBrm and hBrg1 interacting with

components of the 3’end machinery in the nascent RNA by Mass-

spectrometry. We focused in the Drosophila CPSF6 since is known to be a

key regulator of 3’end processing, and observed that CPSF6 the presence of

dBrm significantly increased the binding of CPSF6 to the cleavage sites of

the analysed genes.

29

We propose a mechanism in which SWI/SNF facilitates the recruitment of

the cleavage and polydenylation machinery to the cleavage site of a subset of

genes, and this results in a more efficient 3’ end processing.

30

Study 3

The chromatin-remodelling complex B-WICH is required for

ribosomal transcriptional activation by glucose stimulation.

Aim

Ribosomes are essential for protein production and their genes need to

be expressed according to the cell requirements. It is known that the

expression of ribosomal genes can be affected by different kinds of

stress such as nutrient depravation. The aim of Study 3 is to

investigate the ATP dependent chromatin remodelling complex B-

WICH, comprised of WSTF, SNF2h and nuclear myosin, in the

regulation of Pol I transcribed ribosomal genes upon glucose

stimulation.

Results

In Study 3 we show that B-WICH promotes an open chromatin state at the

Pol I promoter to facilitate the recruitment of factors upon external stimuli.

The B-WICH complex is shown to be required for activating rRNA

transcription upon glucose stimulation since WSTF knock down (KD) cells

failed to activate rRNA transcription after a period of glucose deprivation.

The levels of RNA Pol I at the promoter of 45S were reduced upon glucose

deprivation, and increased upon refeeding, but this did not occur in WSTF

KD cells. We showed that B-WICH increased the accessibility to the

promoter upon glucose stimulation. This made possible the binding of

general transcription factors RRN3 and SL1 to the promoter. Furthermore,

when WSTF was knocked down and the promoter was in an inaccessible

state, the HATs PCAF, GCN5 and p300 were not recruited to the promoter

31

after glucose refeeding and acetylation of H3K9 and H3K14 were not

induced. The knock down of WSTF also reduced the recruitment of known

activator, such as SIRT 7 and c-MYC, to the promoter. To investigate the

underlying mechanism we examined the association with other regulators of

the chromatin state. The NORC complex, comprised of TIP5 and SNF2h,

induces a more permanently silent state characterized by H3K9me3,

heterochromatin proteins and DNA methylation. CHD4, a member of the

NuRD complex, is associated with a poised state, characterized by both

active and silent histone marks and no DNA methylation. What signifies this

state is a positioned nucleosome over the transcription start site, which needs

to be moved to an accessible position further downstream upon activation of

transcription (Xie et al. 2012, Zhao et al. 2016). We investigated the binding

of CHD4 in WSTF KD cells, since B-WICH is not associated with DNA

methylated promoters and we hypothesized that it counteracts a temporally

silent state. CHD4 was present in the promoter under glucose deprivation

and released after glucose refeeding in control cells but not in WSTF KD

cells, where a closed chromatin state was maintained. WSTF and SNF2h

recruitment was not affected in CHD4 KD cells. TTF1, known to recruit

CHD4 to the promoter, was more present at the promoter in WSTF KD cells

while remained low in CHD4 KD cells in both glucose-deprived and

glucose-stimulated cells. We suggest that B-WICH and NuRD complexes

act in a reciprocal mechanism and B-WICH is required for the release of the

NuRD complex upon gene activation. The mechanism of the release is not

fully understood, but our experiments indicate that TTF1, one of the

recruiting factors of NuRD could be involved. Taken together, we propose

that B-WICH is important for establishing an open chromatin state at Pol I

promoter and induces the release of CHD4 to maintain an active permissive

state.

32

3. Conclusions

Study 1 shows that SWI/SNF complexes, more specifically the ATPase

subunits Brg1 and Brm, interact with splicing associated factors and regulate

their recruitment to their target sites, resulting in mRNA alternative spicing

of a subset of exons.

Study 2 shows conserved interactions of Brg1 and Brm with RNA binding

proteins in the nascent RNA. We show that dBrm interact with CPSF6,

promoting the recruitment to the cleavage site of a subset of genes, and

favouring a more efficient 3’end processing.

Study 3 shows that another chromatin remodelling complex, B-WICH, is

important for the regulation of ribosomal genes under glucose starvation

stress. We showed that B-WICH is necessary to induce an open chromatin

state, after a period of glucose starvation followed by glucose refeeding, by

promoting the release of CHD4 (NuRD complex) from the ribosomal genes.

Altogether, these studies show the importance of chromatin remodelling in

the regulation of genes at different steps of their expression. Chromatin

remodelling complexes SWI/SNF and B-WICH are not just limited to open

chromatin to make it accessible to other factors, but also they take part in the

modulation of this recruitment, adding another layer in the regulation of

these genes.

33

4. Acknowledgements

First I would like to thank Anki for giving me the chance of getting a phD,

for all patience during these years and for making a great effort to get it

finished. Second I would like to thank Neus, for recommending me to get

this position and for all the help as a co-supervisor.

Then I would like to thank my current lab mates. Yuan for boosting the

endless several hundred-ChIP experiments and Jaci, for being always helpful

and repeating what you say as many times as I need. Also to former

members, Steffi and Anna, for all help at the beginning of the phD for

adapting to the lab and to Sweden, and Anna R for helping with my

experiments when I was absent.

Of course big thanks in general to my current and former corridor mates, for

all fun during and after working hours. Special thanks to Naveen, Nandu,

Antonio, Andreas for playing badminton at 8 o’clock, Marina for playing

tennis, Mattias for organizing innebandy and Ioanna for helping recovering

from all this activity with your banoffi. Also to Lucas and Sergi for the

company during looong days in the lab and still have something positive to

say.

Big thanks to Neus group members. Andrea, Consuelo, Marta and Simei for

your warm welcome to Sweden and invaluable help during the master’s and

the beginning of the phD in all senses. Judit and Toni for all support in and

out the lab, Shruti, Martín and Jordi for fresh positivism and Patricia for

coming back in the right moment and helping me during the last period.

Finally I would like to give special thanks to Sandra, my supervisor in real

life. For accepting me to start the project of building a family, for coming to

Sweden when seemed cold and sad, and for taking care of Julia when I could

not deal with everything.

34

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DiVA portal1258091/FULLTEXT01.pdf · Två proteinkomplex står i fokus, BRG1 och BRM-innehållande...

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