Current Major Advances in the Regulation of Milk Protein Gene Expression

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Critical Reviews™ in Eukaryotic Gene Expression, 24(4): 357–378 (2014)

1045-4403/14/$35.00 © 2014 Begell House, Inc. www.begellhouse.com

Current Major Advances in the Regulation of MilkProtein Gene Expression

Xi Qian & Feng-Qi Zhao*

Laboratory of Lactation and Metabolic Physiology, Department of Animal Science, University of Vermont,

Burlington, VT

*Address all correspondence to: Feng-Qi Zhao, Department of Animal Science, University of Vermont, 211 Terrill Building, 570 Main St,Burlington, VT 05405; Tel.: (802) 656-0786; Fax: (802) 656-8196; [email protected].

ABSTRACT: During lactation, functionally differentiated mammary epithelial cells convert circulating nutrientsinto various milk components, providing all essential nutrients for the growth and development of mammal neonates.One of the major milk components is milk protein, which includes the casein and whey proteins. Regulation of milk

protein gene expression is dependent on hormonal and developmental cues that modulate the activity of specic

transcription factors and change the chromatin structure in mammary epithelial cells. Understanding the underlyingmechanisms involved in mammary-specic milk protein gene regulation will help improve the yield, quality,

and efciency of milk production and identify important signaling factors and pathways involved in mammarydevelopment, differentiation, lactation, and disease. In this review we rst review advances in the understanding of

the regulatory mechanisms of milk protein genes by hormones, growth factors, and the extracellular matrix, with afocus on transcriptional regulation. We then discuss the relationship between chromatin structure and milk proteingene expression from an epigenetic perspective. Finally, we summarize recent achievements using the mammarygland as a bioreactor for producing pharmaceutical proteins for human use.

KEY WORDS: dairy pharming, epigenetics, gene expression, mammary gland, milk proteins, regulation

ABBREVIATIONS: ATP, adenosine triphosphate; BCE-1, bovine casein enhancer element; BM, basement membrane;C/EBPβ, CAAT/enhancer binding protein β; ECM, extracellular matrix; GC, glucocorticoid; GH, growth hormone;GHR , growth hormone receptor; GR , glucocorticoid receptor; IGF, insulin-like growth factor; INS, insulin; JAK2, Januskinase 2; LAP, liver-enriched transcriptional activator protein; mRNA, messenger RNA; NF-1, nuclear factor 1;Oct-1, octamer binding factor-1; PG, progesterone; PR , progesterone receptor; PRL, prolactin; PRLR , prolactin receptor;

Runx2, runt-related transcription factor 2; STAT5, signal transducer and transcription activator 5; SWI/SNF, Switch/Sucrosenonfermentable; TGF-β, transforming growth factor-β; TSS, transcription start site; WAP, whey acidic protein; YY-1, YinYang 1.

I. INTRODUCTION

Milk is the primary source of nutrients for neo-nates before they are able to consume and digestother types of foods.1 For hundreds of years, dairymilk and other agricultural products derived fromdairy milk (e.g., cheese, butter, and yogurt) have been important foods for humans.2 Among the

many nutrients provided in dairy milk and dairy products, milk protein is an important part of daily protein intake in the human diet. Milk protein is ofhigh biological value to human health; it is a goodsource of essential amino acids, and the aminoacid composition of major milk proteins is well- balanced for use by the human body.3,4

Studies of the regulation of milk protein geneexpression in the mammary gland date back morethan a half century. Knowledge generated fromthese studies has not only provided fundamentalinsight into genetic and nutritional improvement ofmilk composition and milk production but also haselucidated the molecular mechanisms of tissue-specic gene expression. Furthermore, with the

development of genetic engineering technology,the promoters of different milk protein genes have been used to direct the expression of pharmaceuti-cally important proteins in the milk of transgeniclivestock, which is the driving force of the newlyemerging “pharming” industry.



Critical Reviews™ in Eukaryotic Gene Expression

Qian & Zhao358

Studies of the regulation of milk protein geneexpression began as a result of the development ofendocrine organ surgical ablation, access to purehormones for replacement therapy, and the devel-

opment of mammary explant cultures 6 decadesago.5 These early studies demonstrated that basichormone complexes, namely the lactogenic hor-mone prolactin (PRL), glucocorticoids (GCs), andinsulin (INS), synergistically activate milk proteingene expression.6,7 Later studies focused on themolecular pathways involved in the expression ofindividual hormones and the interactions of these pathways. The full view of the molecular detailsof these pathways is emerging: Milk protein geneexpression is regulated at multiple levels withinmammary epithelial cells and depends on con-

certed actions of hormones, local growth factors,cell–cell interactions, and cell–extracellular ma-trix (ECM) interactions that modulate the functionof specic transcription factors, alter cytoskeletal

organization, and change the chromatin state andnuclear structure.

In this review we provide an overview of theregulation of different milk protein genes (mainlytranscriptional regulation) and discuss regulatorymechanisms from the perspective of epigeneticsand chromatin. We mainly focus on data that have

accumulated since 2 previous reviews of this sub- ject published more than a decade ago.8,9

II. MAJOR MILK PROTEINS

Although the proteins in milk can arise from dif-ferent sources, the focus of this review is on themajor proteins that are specically synthesized

in mammary epithelial cells. These mammary-specic proteins in mammals can be grouped into

2 categories: caseins and whey proteins. The pro-teins in cow milk contain ~80% caseins and ~20%

whey proteins, whereas the proteins in human milkcomprise ~40% caseins and ~60% whey proteins.10 Caseins are a family of related phosphoproteins,which include αS1-, αS2-, β-, and κ-caseins. αS1-,

αS2-, and β-casein are characterized as calcium-

sensitive caseins because of their quantitative pre-cipitation with calcium chloride, whereas κ-casein

does not precipitate with calcium chloride and canquantitatively stabilize calcium-sensitive caseins.11 The appropriate amino acid composition of caseinsand their high digestibility make milk essential for

the growth and development of neonates. As a foodsource, they provide not only large amounts of ami-no acids but also phosphorus and calcium. In in-dustry caseins have a wide variety of applications,ranging from being a major component of cheese, being used as a food additive, and being used innonfood applications such as casein-based coatingor sizing agents.12 As a result of their relatively hy-drophobic nature, individual caseins are not verysoluble in aqueous environments; however, caseins

can form multimolecular, spherical casein micellesand thus become colloidally suspended in milk.13

Caseins can be precipitated from milk by reduc-ing the pH to disturb the charge equilibrium in-side colloidal micelles, resulting in a yellow wheysolution. Whey is composed of water, lactose,mammary-specic whey proteins (α-lactalbumin,

β-lactoglobulin, and whey acidic protein [WAP]),

and non-mammary-specic whey proteins (serum

albumin, immunoglobulins, growth factors, etc).Whey proteins have been extensively studied fortheir anti-inammatory and anticancer proper -ties and potential use as a supplementary treat-ment for diseases.14,15 Human mammary-specic

whey proteins contain only α-lactalbumin, mouse

whey proteins contain α-lactalbumin and WAP,

and cow whey proteins contain α-lactalbumin and

β-lactoglobulin. α-Lactalbumin serves as a regula-tory subunit for lactose synthase, and it is criticalfor lactose synthesis.16 β-Lactoglobulin is a major

whey protein in cow milk and an allergen for hu-man infants fed a formula based on cow milk.17 WAP is important in regulating the proliferation ofmammary epithelial cells.18

The genomic location and organization of the

casein and whey protein genes in humans, cattle,and mice are summarized in Tables 1 and 2. Asshown in Table 2, each of the mammary-specic

whey proteins—α-lactalbumin, β-lactoglobulin,

and WAP—is encoded by a single-copy gene witha relatively small size in different chromosomes.In contrast, caseins are encoded by a cluster of dif-



Volume 24, Number 4, 2014

Major Advances in the Regulation of Milk Protein Gene Expression 359

P r o t e i n

G e n e

S y m b o

l *

S p e c i e s

C h r o m o s o m e :

L o c a t i o n *

T r a n s c r i p

t a n d C D S R e g i o n

α S 1 - c a s e i n

C S N 1 S

1

H u m a n

4 : 7 0 , 7

9 6 , 7

9 9 –

7 0 , 8

1 2 , 2

8 9

C a t t l e

6 : 8 7 , 1

4 1 , 5

5 6 –

8 7 , 1

5 9 , 0

9 6

C s n 1 s 1

M o u s e

5 : 8 7 , 6

6 6 , 2

2 4 –

8 7 , 6

8 2 , 5

7 3

β - c a s e i n

C S N 2

H u m a n

4 : 7 0 , 8

2 0 , 9

7 4 –

7 0 , 8

2 6 , 7

2 6

C a t t l e

6 : 8 7 , 1

7 9 , 4

9 9 –

8 7 , 1

8 8 , 0

0 4

C s n 2

M o u s e

5 : 8 7 , 6

9 2 , 6

2 4 –

8 7 , 6

9 9 , 4

2 1

α S 2 - c a s e i n

C S N 1 S

2

C a t t l e

6 : 8 7 , 2

6 2 , 4

5 7 –

8 7 , 2

8 0 , 9

3 6

α S 2 - c a s e i n -

l i k e A

C s n 1 s 2

a

M o u s e

5 : 8 7 , 7

7 4 , 5

6 7 –

8 7 , 7

8 8 , 7

9 7

α S 2 - c a s e i n -

l i k e B

C s n 1 s 2

b

M o u s e

5 : 8 7 8 0 8 1 2 2 –

8 7 8 2 4 4 2 1

κ - C a s e i n

C S N 3

H u m a n

4 : 7 1 , 1

0 8 , 3

3 3 –

7 1 , 1

1 7 , 1

4 5

T A B L E 1 : G e n o m i c

L o c a t i o n a n d O r g a n i z a t i o n o f t h e H u m

a n , C o w , a n d M o u s e C a s e i n G e n e s




Qian & Zhao360

P r o t e i n

G e n e

S y m b o

l *

S p e c i e s


L o c a t i o n *

T r a n s c r i p


κ - C a s e i n

C S N 3

C a t t l e

6 : 8 7 , 3

4 9 , 4

1 0 –

8 7 , 3

8 6 , 9

0 0

8 7 , 3

9 0 , 1

9 7 –

8 7 , 3

9 2 , 7

5 0

C s n 3

M o u s e

5 : 8 7 , 9

2 5 , 6

3 3 –

8 7 , 9

3 2 , 2

6 4

* A d a p t e d f r o m

t h e N C

B I G e n e w e b s i t e

( h t t p : / / w w w . n c b i . n l m . n i h . g o v / p u b m e d ? D b = g e n e & C m d = r e t r i e v e & d o p t = f u l l_ r e p o r t & l i s t_ u i d s = “ g e n e i d ” ; v e r s i o n 1 2 J a n u a r y 2 0 1 2 ) . I n t h e T r a n s c r i p t a n d C D S

R e g i o n

c o l u m n , e a c h v e r t i c a l b a r r e p r e s e n t s a n e x o n , a n d a r r o w s i n d i c a t e t h e o r i e n t a t i o n o f t h e g e n e .

T h e G e n B a

n k a c c e s s i o n n u m b e r f o r e a c h r e f e r e n c e s

e q u e n c e i s

s h o w n .

T A B L E 1 : C o n t i n u e d

P r o t e i n

G e n e

S y m b o

l *

S p e c i e s


L o c a t i o n *

T r a n s c r i p


α - L a c t a l b u m i n

L A L B A

H u m a n

1 2 :

4 8 , 9

6 1 , 4

6 7 –

4 8 , 9

6 3 , 8

2 9

C a t t l e

5 : 3 1 , 3

4 7 , 8

6 1 –

3 1 , 3

4 9 , 8

8 2

L a l b a

M o u s e

1 5 :

9 8 , 4

8 0 , 4

0 0 –

9 8 , 4

8 2 , 6

8 3

β - L a c t o g l o b u l i n

P A E P

C a t t l e

1 1 :

1 0 3 , 3

0 1 , 6

6 4 –

1 0 3 , 3

0 6 , 3

8 1

W h e y a c i d i c

p r o t e i n

W a p

M o u s e

1 1 :

6 , 6

3 5 , 4

8 3 –

6 , 6

3 8 , 6

4 9

* A d a p t e d f r o m

t h e N C

B I G e n e w e b s i t e

( h t t p : / / w w w . n c b i . n l m . n i h . g o v / p u b m e d ? D b = g e n e & C m d = r e t r i e v e & d o p t = f u l l_ r e p o r t & l i s t_ u i d s = “ g e n e i d ” ; v e r s i o n 1 2 J a n u a r y 2 0 1 2 ) . I n t h e T r a n s c r i p t a n d C D S

R e g i o n

c o l u m n , e a c h v e r t i c a l b a r r e p r e s e n t s a n e x o n , a n d a r r o w s i n d i c a t e t h e o r i e n t a t i o n o f t h e g e n e .

T h e G e n B a

n k a c c e s s i o n n u m b e r f o r e a c h r e f e r e n c e s

e q u e n c e i s

s h o w n .

T A B L E 2 : G e n o m i c

L o c a t i o n a n d O r g a n i z a t i o n o f t h e H u m

a n , C a t t l e , M o u s e W h e y P r o t e i n G e n e s





ferent casein genes on the same chromosome. Inhumans the αS1-, β-, and κ-casein genes are se-quentially clustered in chromosome 4 (Table 1).

In comparison with the human casein gene locus,

there is one αS2-casein gene in cows and two re-lated αS2-casein genes (αS2-casein-like A and

αS2-casein-like B) in mice (Table 1). The genes

encoding the calcium-sensitive caseins (αS1-, β-,

and αS2-casein genes) are evolutionarily related,

whereas the κ-casein gene is not derived from a

common ancestral gene, although its expression prole is similar to other casein genes.19

III. TRANSCRIPTIONAL REGULATION OFMILK PROTEIN GENE EXPRESSION

Transcriptional regulation of milk protein geneexpression is regulated by cis-regulatory regions(promoter and/or enhancer). The cis-regulatoryregion is a stretch of DNA (100–1000 bp) withtranscription factor binding sites that are clusteredinto modular structures. Through protein–DNAand protein–protein interactions, these modularlystructured regions integrate positive and negativeregulatory signal transduction pathways induced by various extracellular stimuli to regulate geneexpression by controlling the initiation and/or stabilization of the transcription complex on gene promoters and enhancers.9 Previous studies usingstable or transiently transfected mammary/non-mammary cells and transgenic mice establishedthe functional importance of various transcriptionfactors in the regulation of milk protein genes.9 None of these transcription factors are specic to

the mammary gland. Therefore it is the specic

combination of these transcription factors thatleads to the unique temporal and spatial expression

proles of milk protein genes.

The β-casein and WAP gene promoters have

been extensively studied for decades as models forhormone signaling control of milk protein geneexpression. β-Casein gene regulation involves

two principal cis-regulatory regions, a proximal promoter and a distal enhancer. The core proxi-mal promoter, homologous in humans, cows, androdents, extends ~250 bp upstream from the tran-

scription start site (TSS) of the β-casein gene, and

the evolutionally conserved enhancer is located between −1.6 and −6 kb upstream of the 5′ region

of the TSS in different mammalian species19,20

(Fig. 1A). The hormone-responsive β-casein prox-imal promoter has so-called lactogenic responseelements that harbor multiple or a single bindingsite(s) for transcription factors, mainly includ-ing signal transducer and transcription activator 5(STAT5),22,47 GC receptor (GR),23,48 CAAT/enhanc-er binding protein β (C/EBPβ),24,49 octamer bind-ing factor-1 (Oct-1),36,50 runt-related transcriptionfactor 2 (Runx2),37 and the repressive transcriptionfactor Yin Yang 1 (YY-1).25,26 The distal enhancerof the β-casein gene, also known as the ECM-re-sponsive element, is responsive to ECM and lacto-

genic hormones and contains recognition sites forC/EBPβ and STAT5.20,21,51

The WAP gene promoter also contains 2 regu-latory regions, one proximal (−50 to −150 bp in

both the rat and mouse) and one distal to the TSS(−720 to −820 bp in the rat and −530 to −630 bp

in the mouse).42,52 Both of these regions containconsensus binding sequences for a number of tran-scription factors, including nuclear factor 1 (NF-1),GR, and STAT5, which have been demonstrated to be responsible for mammary-specic WAP gene

expression.42–44

IV. HORMONAL REGULATION OF MILKPROTEIN GENES

A. PRL and GC

Long before the discovery of the molecular mecha-nisms of the activation of milk protein gene expression, researchers demonstrated the synergism between PRL and GC.6,7 The most extensivelystudied milk protein gene is the β-casein gene. Us-

ing transgenic mouse models and cell culture sys-tems including primary or transformed mammaryepithelial cells, researchers established in the 1990sthat STAT5, GR, and C/EBPβ are important signal

transducers that mediate PRL and GC synergism inthe induction of β-casein gene expression.9,24,27,53–55 STAT5, as well as its 2 closely related protein iso-




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types, STAT5A and STAT5B, is the leading tran-scription factor responsible for PRL signaling.56 The binding of PRL to the PRL receptor (PRLR) trig-

gers activation of Janus kinase 2 (JAK2). ActivatedJAK2 phosphorylates tyrosine residues on the PRLRand creates docking sites for Src homology 2 (SH2)

FIG. 1: Schematic representation of the cis-regulatory regions of milk protein genes. DNA binding sites for tran-

scription factors are shown as different shapes. A. Transcription factor binding sites mapped in the promoter andenhancer of the β-casein gene. The β-casein gene enhancer was originally identied in the bovine species; thus

it was named bovine casein enhancer element (BCE-1).21 These binding sites are highly conserved in β-casein

gene promoters and enhancers in rabbits, rats, mice, goats, sheep, and cows.20 Signal transducer and activator of

transcription 5 (STAT5), CCAAT/enhancer binding protein (C/EBP), and Yin Yang 1 (YY-1) binding sites and half glu -

cocorticoid response elements (½ GREs) for glucocorticoid receptor in the β-casein proximal promoter and/or distal

enhancer have been functionally veried and extensively studied.21–35 The binding sites for octamer factors (Oct) and

runt-related transcription factor 2 (Runx2) have recently been characterized. 36–40 The E26 transformation-specic

(Ets) site and nuclear factor 1 (NF-1) binding site are predicted based on sequence identity.20 B. Transcription fac-

tor binding sites mapped in the promoter of the αS1-casein gene promoter. These binding sites are putative based

on sequence identity, and they are highly conserved in αS1-casein gene promoters in buffalo, yak, cows, sheep,

goats, camels, and humans.41 C. Structural organization of the transcription binding sites in the 2 highly conserved

cis-regulatory regions of the rodent whey acidic protein (WAP) promoters. These binding sites are highly conserved

in rats and mice.42 NF-1 and STAT5 binding sites and ½ GREs have been functionally characterized in rats and/or

mice.35,42–44 D. Schematic representation of the transcription factor binding sites in the β-lactoglobulin proximal pro-moter. STAT5, NF-1, and activator protein 2 (AP-2) sites have been veried in the β-lactoglobulin gene promoters of

sheep and/or cows.32,45,46 The numbers indicate positions relative to the transcription start sites (TSSs, +1).





domain–containing proteins. Src homology 2–con-taining STAT5 is then recruited and phosphorylated by JAK2 at a conserved tyrosine residue within thecarboxyl terminal transcriptional activation domain

(Y694 for STAT5A and Y699 for STAT5B).28,29

Phosphorylated STAT5 dimerizes, translocates intothe nucleus, and induces β-casein gene transcription

by binding to clustered STAT5 binding sites in both promoters and enhancers. Of the 2 STAT5 proteinisotypes, STAT5A is the principal and indispensablemediator of mammary epithelial cell differentiationand milk protein gene expression, whereas STAT5Bis more important for growth hormone (GH) sig-naling in the liver.57,58 GC alone can barely activateβ-casein gene expression; however, GC potentiates

PRL signaling through synergistic protein–protein

interactions between GR and STAT5, leading tomuch more robust induction than PRL alone.23,48,59 AGC potentiation effect occurs in a half GC responseelement binding-dependent or -independent man-ner.23,60–62 The interaction between GR and STAT5 promotes sustained STAT5 tyrosine phosphoryla-tion and STAT5–DNA binding.63 C/EBPβ is another

transcription factor that binds to the β-casein proxi-mal promoter and enhancer as a homo- or heterodi-mer in response to PRL and/or GC,24,30,49 and it isimplicated in the regulation of milk protein gene expression.24,27 Three C/EBPβ protein isotypes, which

are translated from a single C/EBPβ messenger RNA

(mRNA), have been identied, including 2 tran-scription-activating isoforms called liver-enrichedtranscriptional activator proteins (LAP and LAP2)and one inhibitory isoform, the liver-enriched tran-scriptional inhibitory protein.31,49,64 The LAP C/EBPβ isoform synergizes with STAT5 and GR dur -ing the induction of β-casein gene expression by

PRL and GC in a reconstituted COS-7 cell system.59 The cooperative effects of STAT5 and C/EBPβ for

PRL- and GC-induced β-casein gene transcription

are mediated by the GR.59

All of these transcriptionfactors (i.e., STAT5, GR, and C/EBPβ) interact with

the p300 coactivator, which remodels the chromatinconrmation via its intrinsic histone acetyltransfer -ase activity to facilitate gene transcription.65–67 Inaddition to these positive regulatory transcriptionfactors, YY-1 constitutively binds the β-casein gene

proximal promoter in the absence of lactogenic hor-mones and represses β-casein gene expression.25,26 The YY-1 site in the β-casein gene proximal pro-moter is a low-afnity site, and in response to PRL

its association can readily be interrupted by STAT5and C/EBPβ binding at adjacent sites.25,30

In addition to the transcription factor bindingsites mentioned above, there are 2 highly conservedadjacent binding sites for Oct-1 (36) and Runx2in casein gene proximal promoters37 (Fig. 1). BothOct-1 and Runx2 bind, both in vivo and in vitro, tothe endogenous β-casein gene promoter in mam-mary epithelial cells.36–39,68 Oct-1 alone can activate both basal and hormonally induced β-casein gene

promoter activity,38 whereas Runx2 alone cannot.37 Runx2 cooperates with Oct-1, however, leading tohigher activation of the β-casein promoter than oc-curs with Oct-1 alone.37 This cooperation may beexplained by the fact that Oct-1 stimulates the re-cruitment of Runx2 to the β-casein gene promoter

by physically interacting with Runx2.37 In additionto Runx2, Oct-1 synergizes with STAT5 and GRthrough physical interaction to activate β-casein

gene expression in response to PRL and GC.39 Similar to its stimulating effects on Runx2 recruit-ment, Oct-1 enhances or stabilizes the binding ofSTAT5 and GR to the β-casein promoter in re-

sponse to PRL and GC.

39

In addition to the combi-nation of PRL and GC, Oct-1 binding activity canalso be induced by progesterone (PG),36 which is areproductive hormone that inhibits β-casein gene

expression.69 Oct-1 has been shown to interact withthe PG receptor (PR)70; therefore it is possible thatOct-1 also participates in the repression of β-casein

gene expression via interaction with different tran-scription factors such as PR.

PRL and GC also regulate milk protein geneexpression at other levels, for example, stabili-zation of milk protein mRNA and milk protein

mRNA translation, which have been well re-viewed elsewhere.71

B. Insulin

The functional role of INS in regulating milk pro-tein production is supported by in vitro and in vivo




Qian & Zhao364

studies. Early in vitro studies of mouse, rat, and bovine mammary explant cultures showed that, inaddition to maintaining mammary tissue in cul-ture, INS is required for the maximum induction

of the major casein and whey milk protein genes by PRL and hydrocortisone (a type of GC).72–74 Inin vivo studies of cows using a hyperinsulinemic-euglycemic clamp approach in which circulatingINS concentrations were elevated 4-fold while eu-glycemia was maintained via the infusion of ex-ogenous glucose, milk protein yield was increased by 15% within 4 days of treatment with a hyperin-sulinemic-euglycemic clamp.75,76 Because the ad-ministration of extra glucose to well-fed cows doesnot increase milk yield, and because INS does notinduce an acute increase in glucose uptake by ru-

minant mammary glands,77–81 the increase in milk protein yield possibly resulted from a stimulatoryeffect of INS on mammary epithelial cells.

The underlying molecular mechanism of howINS regulates milk protein synthesis is not well un-derstood. However, previous studies suggest thatINS may play an important role in milk proteinsynthesis at multiple levels. First, at the transcrip-tional level, INS may synergistically cooperatewith PRL and hydrocortisone to induce milk pro-tein gene transcription by stimulating the expres-sion of E74-like factor 5 (Elf5) via phosphoinosit-ide 3-kinase/Akt signaling,82,83 as demonstrated in bovine and murine mammary explant cultures.84,85 Elf5 is a transcription factor that belongs to theE26 transformation-specic (Ets) family, and it

regulates mammary epithelium proliferation anddifferentiation and the transcription of milk pro-tein genes.86–89 INS-dependent milk protein genetranscription may also be accomplished by INS-induced expression and the activity of STAT5.9,84 Second, in CID-9 mammary epithelial cells, whichwere derived the COMMA-D cells originally iso-

lated from the mammary glands of mice in midpregnancy,90 INS alone or INS plus PRL increasesthe rate of milk protein mRNA translation, where-as PRL alone has no effect.91 INS enhances thetranslation of β-casein by increasing the initiation

of translation and lengthening the mRNA poly(A)tail by cytoplasmic polyadenylation element bind-

ing proteins.91 Finally, more recent studies of cowsand mice suggested that INS may enhance proteinsynthesis by stimulating genes involved in folatemetabolism.84,85,92 Folate metabolism plays an im-

portant role in protein synthesis by accepting andreleasing 1-carbon units, which is also known asthe 1-carbon pool. A functional role of folate inmilk protein synthesis is supported by the fact thatfolate supplementation in lactating cows results ina signicant increase in milk production and milk

protein yield.93–95

C. Progesterone

PG is a steroid hormone secreted by the corpusluteum. PG exerts its primary action through the

nuclear PR. When bound with PG, the PR dissoci-ates from protein chaperones, dimerizes, and bindsto specic binding sites in its target genes, regu-lating their expression by recruiting its coactiva-tors.96 Circulating concentrations of PG increasethroughout pregnancy, followed by a rapid declineat parturition. In early pregnancy, PG promotesmammary gland development and functional dif-ferentiation. In mid- to late pregnancy, PG inhibitsthe production of milk proteins and the closing ofthe tight junctions until parturition. At parturition,a dramatic decline in circulating PG, in combina-tion with decreased PR expression in the mammarygland, results in tight junction closure and copious production of milk protein.97–99 The repressive ef-fects of PG on milk protein gene expression wereinitially observed in an experiment in which ovari-ectomy led to transient lactogenesis, which is char-acterized by the transcription of important milk protein genes such as caseins and α-lactalbumin in

mice during late pregnancy; PG, but not other hor-mones (estrogen, PRL, or GC), abolishes the tran-sient lactogenesis triggered by ovariectomy.100,101

Only recently have we begun to understandthe molecular mechanism of the repression ofmilk protein gene expression by PG. Using differ-ent cell culture systems reconstituted to expressthe PR, Buser and colleagues69 found that directantagonism between activated PR and STAT5/GR signaling contributes to the physiological role





of PG in repressing lactogenic hormone–inducedβ-casein transcription in mammary epithelial

cells. However, direct transcriptional repressionof milk protein genes by PR is unlikely to be the

primary mechanism for PG repression in mamma-ry epithelial cells during pregnancy because PR isexpressed only in a scattered subset of mammaryepithelial cells during pregnancy.99,102 It is possiblethat the inhibitory effects might be mediated by paracrine factors regulated by PG.103 Transform-ing growth factor-β (TGF-β) is likely to be the me-diator of PG action in repressing milk protein geneexpression because of several lines of evidence:(1) TGF-β antagonizes PRL-induced signals in

mammary epithelial cells and inhibits alveolar for-mation and the synthesis of milk proteins during

pregnancy.104–111 (2) The expression prole of theTGF-β isoforms TGF-β1, TGF-β2, and TGF-β3 in

the mammary gland correlates with changes in theconcentration of plasma PG during the transitionfrom pregnancy to lactation.107,108 (3) In bovinemammary cells and tissue, PG tends to increaseTGF-β expression.112,113 Moreover, PG signicant-ly upregulates TGF-β expression in normal human

osteoblast-like cells.114

Furthermore, milk protein genes are alreadytranscribed at a considerable level during mid- to

late pregnancy, when PG concentrations are stillhigh,115 implying that PG may mainly inhibit milk protein synthesis at the post-transcriptional levelor milk protein secretion.

D. GH and Insulin-like Growth Factor-1

GH, also known as somatotropin, is a peptide hor-mone synthesized and secreted by the anterior pi-tuitary. GH stimulates cell growth and changes in protein, carbohydrate, and fat metabolism. Upon binding to its cell membrane–integrated GH recep-

tor (GHR) in target tissues, GH activates variousintracellular signaling molecules to regulate geneexpression and protein modications.116 One well-dened example of GH function is the stimula-tion of insulin-like growth factor (IGF)-1 expres-sion.117,118 IGF-1 is believed to mediate many of thegrowth-stimulating and metabolic effects of GH.

GH is well known for its galactopoietic effectson the mammary gland.118,119 However, the under-lying mechanisms mediating the effects of GH on protein synthesis remain unclear. Early studies us-

ing ligand binding assays failed to detect the GHRin bovine mammary glands,120,121 leading to thewidely accepted hypothesis that GH acts on themammary gland through IGF-1 produced locallyand by the liver.122 The direct effects of IGF-1 inthe mammary gland are supported by the fact thatlocally increased IGF-1, either by local arterialIGF-1 infusion in the mammary gland (in lactatinggoats) or mammary-targeted IGF-1 expression (inmice),123,124 increases milk production. This resultmay be attributable to the ability of IGF to inhibitapoptosis in the mammary gland and stimulate

mammary epithelial cell proliferation and glucosetransport, as occurs in cows.125,126 However, over-expression of mammary-specic IGF-1 in swine

and rabbits did not impact milk production andcomposition.127,128 The exact role of IGF in GH-enhanced milk yield requires further investigation.

While it is widely accepted that GH has onlyindirect action on mammary gland function, the di-rect role of GH is emerging. Recent studies showedthat both GHR mRNA and protein are expressedin the stromal and epithelial tissues of the bovine

mammary gland.

129–131

GHR protein expression inlactating mammary glands was higher than that innonlactating mammary glands.129 In addition, GHis capable of stimulating the mRNA expression ofmilk protein genes in bovine mammary epithelialcell lines, such as MAC-T and BMEC, and bovinemammary explant cultures.132–136 Furthermore, asrecently demonstrated, in bovine and swine mam-mary gland tissues GH may upregulate milk pro-tein mRNA translation initiation and elongation viathe mammalian target of rapamycin pathway.137–141 Thus GH may directly stimulate milk protein syn-

thesis at the transcriptional and translational levels.

V. EXTRACELLULAR MATRIX

The ECM is a group of lamentous and insoluble

proteins that are present between clusters of cellsin all tissues.142 In addition to providing tensile




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support, the ECM provides channels for the com-munication between cells in a given tissue. TheECM is categorized into 2 types: the stromal ECMand the basement membrane (BM).142 The stromal

ECM resides in connective tissues, whereas theBM, also known as the basal lamina, separates theepithelium from the stroma in any given tissue.Representative BM constituents include laminin,type IV collagen, nidogen/entactin, and heparinsulfate.143 The ECM can alter gene expression pro-les by inuencing cell morphology and nuclear

and chromatin organization.142,144 The ECM per-forms its function by binding to integrin receptorson the cell surface and initiating mechanical andchemical signaling.142

The ECM cooperates with soluble cues, in-

cluding hormones and growth factors, to guidemammary gland development, functional differ-entiation, alveolar morphogenesis, lactation, andinvolution. During lactation, the ECM is neededto induce milk protein gene expression. With theexception of mammary epithelial cell lines such asHC11, which can deposit its own laminin matrixafter reaching conuence, for primary mammary

epithelial cells and most other mammary epithelialcell lines endogenous milk protein gene expressioncan be induced by lactogenic hormones only whenthey are cultured on a laminin-rich ECM.9 The ECMis required for the induction of milk genes, such as

caseins, β-lactoglobulin, and WAP, in response to

lactogenic hormones.145–148 The ECM-dependentregulation of milk protein genes is mediated bycertain DNA sequence elements in their promoter

regions. For example, promoter truncation analysisrevealed an ECM-responsive element, originallynamed bovine casein enhancer element (BCE-1),~1.5 kb upstream of the bovine β-casein TSS.21 BCE-1 was highly conserved among different spe-cies and was dened as a β-casein distal enhancer

(Fig. 1A) that contains binding sites for variousmammary transcription factors.19–21 The β-casein

proximal promoter was more recently found to beresponsive to the ECM as well.146 ECM-responsiveelements in other milk protein genes and othergenes are summarized elsewhere.144 However, ex-actly how the ECM regulates milk protein genes

via these ECM-responsive DNA elements is notwell understood. Current evidence suggests sev-eral potential mechanisms.

One potential mechanism by which the ECM

activates milk protein gene expression is by in-ducing the binding of mammary transcriptionfactors to ECM-responsive DNA elements. Us-ing primary mammary epithelial cells from micein midpregnancy, Streuli et al.147 demonstratedthat, in the presence of PRL, only cells culturedon laminin-rich ECM—but not cells cultured oncollagen I—are capable of inducing STAT5 DNA binding activity. These authors also found that theDNA binding activity of NF-1 is stimulated onlyin cells cultured on a laminin-rich ECM, but thisactivation occurs independent of PRL, whereas the

DNA binding activity of specicity protein 1 (sp1)is induced in cells cultured on plastic or collagenI substrata in the presence of PRL.147 These datasuggest that the activities of transcription factorsin mammary epithelial cells are differentially regu-lated by the type of substratum on which the cellsare cultured. Subsequently, Edwards et al.149 foundthat exposure to a laminin-rich ECM is required

for the PRL-induced phosphorylation and translo-cation of STAT5 into the nucleus in primary mam-mary epithelial cells. In EpH4 cells, which wereoriginally isolated from the mammary tissue of aBalb/c mouse in midpregnancy,150 PRL can inducethe transient phosphorylation of STAT5 indepen-dent of a laminin-rich ECM, but sustained STAT5activation, which is necessary for the induction ofβ-casein transcription, depends on the presence of

a laminin-rich ECM.151 These results suggest thatcrosstalk between the ECM and the PRL signal-ing pathway is needed to induce milk protein geneexpression. In support of this hypothesis, evidenceshows that the ECM cooperates with PRL to in-duce the binding of STAT5, C/EBP-β, and RNA

polymerase II to casein gene promoters, whereaseither alone fails to do so.146

A second potential mechanism is that media-tion of milk protein gene transcription by ECM-responsive DNA elements is involved in changesin chromatin structure. Studies using the CID-9mammary epithelial cell line revealed that BCE-1





must be stably integrated into the genome to be-come activated by the ECM.21 Furthermore, inhibi-tors of histone deacetylase were sufcient for stim-ulating the activity of chromatin-integrated BCE-1

in the absence of ECM.21

Together, these resultssuggest that epigenetic mechanisms and chromatinremodeling are involved in the ECM-mediated in-duction of milk protein gene transcription, whichis discussed in the next section.

In addition to regulation through ECM-re-sponsive DNA elements, the ECM can initiatemechanical signals that can induce changes in cellshape and cytoskeletal organization, which may be crucial for the hormonal induction of milk pro-tein genes. For example, when EpH4 cells werecultured in a laminin-rich ECM, they formed into

polar, acinar-like structures, and their cytoskeletonreorganized into a cortical network that is required

for PRLR/STAT5 signaling.151–153 During these processes, exposure of mammary epithelial cellsto a laminin-rich ECM recruits PRLR to the basalsurface of acini, allowing for the binding of PRLand thus the activation of PRLR/STAT5 signal-ing.151 Furthermore, increased ECM stiffness canreduce β-casein expression by regulating cellular

actin polymerization, implying that matrix compli-ance is required for cytoskeleton reorganization, a

crucial factor for cell-specic gene expression.

154

VI. EPIGENETICS AND CHROMATINSTRUCTURE IN THE REGULATION OF MILKPROTEIN GENE EXPRESSION

Casein and WAP gene expression is regarded as amarker for the functional differentiation of mam-mary epithelial cells, and epigenetic mechanisms playing a key role in mammary gland develop-ment and functional differentiation have beensuggested.155 It is therefore conceivable that epi-

genetic mechanisms are implicated in the tissue-and developmental stage–specic regulation of

milk protein genes. Epigenetics refers to the studyof heritable changes in genome function that oc-cur due to chemical changes in DNA and its sur-rounding chromatin rather than changes in DNAsequences. Epigenetic regulation is mediated by

DNA methylation, histone modications (acetyla-tion, ubiquitination, methylation, and phosphory-lation), and microRNAs, which modulate the chro-matin conrmation and thus gene expression. The

development of new technologies, such as chroma-tin immunoprecipitation followed by quantitative

polymerase chain reaction, microarrays, and, morerecently, DNA sequencing, and the availability of

complete genomic sequences enable epigenetic

modications at specic genomic sites as well as

on a global scale to be quantitatively analyzed.

Studies investigating epigenetic mechanisms in ro-dent mammary gland development and functionaldifferentiation and dairy cow milk productionhave recently been reviewed elsewhere.155–157 Herewe review only the involvement of epigenetic and

chromatin mechanisms that regulate milk proteingene expression.

A. DNA Methylation

DNA methylation refers to the conversion of thecytosine bases in DNA strands into 5-methylcy-tosine. DNA methylation results in the repressionof gene expression, perhaps by blocking cis-reg-ulating elements where transcription activatorsshould bind.158 An inverse correlation betweenDNA methylation status and milk protein geneexpression has been documented in many studies.Approximately 4 decades ago, Johnson et al.159 no-ticed that certain restriction sites in the rat β- and

γ-casein genes from lactating mammary glands

are readily digested by the methylation-sensitiverestriction enzymes MSpI and HpaII , but DNAsamples from liver are resistant to digestion at thesame restriction sites. Using a similar strategy, therat κ-casein gene was hypomethylated in lactat-ing mammary glands, but it was hypermethylatedin nonmammary tissues and nonlactating mam-

mary glands.160

The same study also implied the possibility that lactogenic hormonal induction ofκ-casein gene expression is mediated by reducing

DNA methylation.160 Hypomethylation during lac-tation has also been described for 3 specic sites

anking the bovine αS1-casein gene, and meth-ylation of 1 of these 3 sites is inversely correlated




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with αS1-casein gene expression.161 In addition tothe casein locus, the WAP gene is also specically

hypomethylated in the lactating mammary glandin its coding and 5′ anking regions, including the

proximal promoter and a hormone-responsive dis-tal site.162,163 The relationship between methylationand bovine αS1-casein gene expression during dif -ferent physiological states and during mastitis has been explored more recently.164,165 DNA methyla-tion at a STAT5-binding enhancer located −10 kb

upstream of the TSS can be induced following an18-hour nonmilking period, and this induced meth-ylation occurs before the decline in PRL signalingand milk protein gene expression that takes place at24 to 36 hours after milking.156,164 Mastitis caused by Escherichia coli infection of the udder results

in DNA methylation in the same region that is as-sociated with αS1-casein gene silencing.165 Thusmilk protein gene expression is potentially regu-lated by DNA methylation, which is inuenced by

cues not only from mammary gland developmentand functional differentiation but also from physi-ological circumstances and health status. However,how these cues change the DNA methylation sta-tus and how the modied DNA methylation status

regulates milk protein gene expression remain un-answered.

B. Histone Modifcations

Histone modications also have been implicated in

cell differentiation and the transcriptional controlof tissue-specic and inducible genes. Histone-

modifying complexes catalyze the addition or re-moval of various chemical elements on histones.These enzymatic modications include acetyla-tion, methylation, phosphorylation, and ubiquiti-nation and primarily occur at N-terminal histonetails. Some modications affect the binding af -

nity between histones and DNA and tighten thecondensed DNA wrapped around histones, pre-venting the binding of transcription factors to DNAand leading to gene repression. By contrast, his-tone acetylation relaxes chromatin condensationand exposes DNA for transcription factor binding,leading to increased gene expression.

Emerging evidence implies that histone modi-cations are involved in the regulation of milk pro-tein gene expression. Using chromatin immunoprecipitation, enrichment in histone H3 acetylation

at proximal promoters and many potential distalregulatory elements in the mouse casein and WAPgene loci occurs specically in lactating mammary

glands but not in the liver.19,155 Furthermore, at thecellular level, lactogenic hormones recruited thehistone-modifying enzyme p300 to the β-casein

promoter in HC11 mouse mammary epithelialcells, which correlated with an increase in histoneH3 acetylation and the stable association of RNA polymerase II at promoters and enhancers.30 Threeclassical milk transcription factors—STAT5, GR,and C/EBPβ—can interact with p300.65–67 Histone

acetylation presumably contributes to mammary-specic milk protein gene transcription.

By contrast, the ECM and ECM-induced cellshape changes lead to a global deacetylation of his-tones H3 and H4 and a global reduction in geneexpression in HMT-3522-S1 and HMT-3522-T4-2human mammary epithelial cells.166 However,these ndings do not rule out the possibility that

there are locally hyperacetylated regions involvedin tissue-specic gene expression during cell dif -ferentiation. For example, the ECM alone or in

combination with PRL induces histone acetylationat the promoters of the αs1-casein and β-casein

genes in primary rabbit mammary epithelial cellsand EpH4 mouse mammary epithelial cells.146,167

C. Chromatin Structure

More than a decade ago, researchers noticed thatmethylated CpG islands coincide with hypoacet-yla histones.168 Subsequent studies showed that

methylated DNA can be bound by methyl-CpG binding domain proteins, which in turn recruit ad-

ditional proteins, such as histone deacetylases andother chromatin remodeling proteins that modifyhistones, thereby forming a compact, closed chro-matin structure called heterochromatin.169–172 DN-ase I digestion has been widely used to identifyopen chromatin regions by the presence of DNaseI hypersensitive sites. Using this method, DNase I





hypersensitive regions have been identied in lac-tating mammary glands at sites of different milk protein genes, such as ovine β-lactoglobulin; rat,

rabbit, and mouse WAP genes; and mouse casein

genes.43,44,155,173–175

The DNase I hypersensitive sitesare usually located upstream of milk protein genes,overlap with regions with DNA hypomethylationand positive histone marks,155 and correlate withtranscription factor binding sites, such as bindingsites for STAT5, GR, and NF-1.43,44,176,177 Severallines of evidence indicate that the DNase I hyper-sensitive sites of milk protein genes seem to be de-velopmentally regulated and potentially activated by lactogenic hormones, as indicated by in vivo

and in vitro experiments.174,177,178 This possibilityis supported by a recent study using DNase I hy-

persensitivity, histone H3 acetylation enrichment,and H3K4 dimethylation enrichment as indicatorsof open chromatin; researchers found that milk protein gene loci progressively gain positive chro-matin marks from puberty to lactation in conjunc-tion with mouse mammary gland development anddifferentiation.179 For example, distal regulatoryregions within casein gene loci and the WAP generegion present open chromatin marks after puber-tal development, and these open chromatin marks persist after lactation ceases, whereas proximal promoters gain only an open chromatin conforma-tion during pregnancy and close at the weaningstage.179 These results suggest a model in whichmilk protein gene loci achieve a chromatin struc-ture during pubertal development that is poised to be sensitive to lactogenic hormones to achieve thelactation capacity of the mammary gland.

Chromatin structure is inuenced by not

only histone modications but also adenosine

triphosphate (ATP)–dependent remodeling. ATP-dependent chromatin remodeling complexes havea common ATPase domain, and energy from the

hydrolysis of ATP allows these remodeling complexes to reposition (slide, twist, or loop) nucleo-somes along DNA, expelling histones from DNAor facilitating the exchange of histone variants,thus creating nucleosome-free DNA regions forgene activation.180 ATP-dependent chromatin re-modeling Switch/Sucrose nonfermentable (SWI/

SNF) complexes are implicated in cellular dif-ferentiation and tissue-specic gene transcrip-tion.181,182 Recruitment of SWI/SNF complexesto the cis-elements of tissue-specic genes is

mediated by association with specic transcrip-tion factors, such as GR and C/EBPβ, or binding

to acetylated histone tails through their bromo-domains.183–186 For milk protein gene regulation,Xu et al.146 showed that laminin-rich ECM andPRL cooperate to recruit the SWI/SNF complexto β-casein and γ-casein promoters via interaction

with GR, STAT5, and C/EBPβ, which are needed

for stable RNA polymerase II binding and genetranscription. Thus, ECM and PRL may be able toregulate casein gene transcription via ATP-depen-dent chromatin remodeling.

In addition to the biochemical level, chro-matin conformation changes, such as chromatin bending and looping, can occur on a macroscop-ic scale and lead to the interaction of distantlyspaced genomic regions.187,188 This possible high-order interaction provides a way for transcrip-tion factors and other coactivators, associated at proximal promoters and distal enhancers, to co-operate with each other through chromatin loop-ing-mediated protein–protein interaction. Lacto-genic hormones promoting physical interaction

between the β-casein gene proximal promoterand an upstream enhancer in HC11 cells and pri-mary 3-dimensional mammary acini cultures has been demonstrated.189 This interaction is blocked by PG-induced PR binding to the promoter.189 Furthermore, developmental regulation of DNAlooping between β-casein regulatory regions was

observed in lactating but not virgin mouse mam-mary glands, and the DNA looping was directlycorrelated with β-casein gene transcription.155

VII. EXPRESSION OF TRANSGENES IN THE

MAMMARY GLANDS OF TRANSGENICANIMALS

A number of transgenic animals harboring trans-genes containing the 5′ and 3′ anking sequences

of milk protein genes have been generated. Theseanimals have been used not only for determining




Qian & Zhao370

the functional importance of cis elements in milk protein gene regulation but also for producing pharmaceutical proteins in the milk of these trans-genic animals. Producing pharmaceutical proteins

in animal mammary glands has led to the devel-opment of a new eld of biotechnology known as

mammary bioreactor.Before the development of animal bioreac-

tors, pharmaceutical proteins were either extractedfrom plants and animals or produced in bacterialor mammalian cell cultures. The transgenic animal bioreactor is superior because of its scalability.190 Because transgenic animals can transmit trans-genes to their offspring, productivity can easily be increased by optimizing the efciency of the

breeding program. The large-scale production of

proteins by cell culture is, however, expensiveand time-consuming. The cost of building a mod-ern cell culture–based industrial bioreactor is over$100 million, and it may take several years to buildsuch a facility. Another appeal of genetically engi-neered animals is the ability to induce many post-translational modications, such as disulde bond

formation, tyrosine sulfation, glycosylation, andcarboxylation, and proper folding of expressed pro-teins, which occurs in native cells and is required

for their biological activity.191 These features aredistinctly superior compared with producing re-combinant proteins in prokaryotes such as bacteria because bacteria lack the post-translational ma-chinery found in mammalian cells. In addition, re-combinant proteins synthesized in bacteria cannot be secreted into an extracellular environment butoften accumulate as insoluble aggregates in inclu-sion bodies.191 Producing proteins by mammaliancell culture can overcome the shortages of pro-karyotes systems; however, mammalian cells usu-ally require the addition of serum to culture media,

and serum may be contaminated by unknown or

undetected viruses.190

Furthermore, expression ofrecombinant proteins in the mammary gland offersmore advantages.192 The sole function of the mam-mary gland is to produce milk, which is composedof up to 4% protein (40 g protein/L). An averagedairy cow produces approximately 40 kg milk/day,with up to 1.6 kg of proteins secreted each day.

Thus the mammary gland is a natural protein-se-creting organ with high capacity. In addition, milkhas only a few main protein components. Thus re-combinant proteins expressed in milk are relative-

ly easier to extract. Extensive studies have shownthat the mammary gland has the ability to synthe-size, properly fold, assemble, and secret complex proteins.191,192 These unique properties make the

mammary gland the best available bioreactor.Genetically engineered mammary glands as

animal bioreactors have mainly focused on pro-ducing biopharmaceuticals. Many different biopharmaceuticals, such as human recombinanterythropoietin, human coagulation/clotting factorsVIII and IX, and human α-1-antitrypsin, have been

produced in the milk of different transgenic mam-

mals.193–196 In 2006 the European Commission ap- proved a biopharmaceutical protein produced inthe milk of goats, antithrombin III (commerciallynamed Atryn), for the treatment of patients with he-reditary antithrombin deciency.197 Atryn was thenapproved by the US Food and Drug Administrationin 2009.197 Atryn was the rst-ever pharmaceutical

protein produced in the milk of transgenic animalsand the rst recombinant antithrombin product ap- proved worldwide. In our laboratory, transgenicmice were recently generated to harbor the human proinsulin gene driven by a goat β-casein gene pro-moter.198 These animals secrete large concentra-tions of proinsulin in their milk without apparenthealth effects, and the mature insulin derived fromthe milk proinsulin retains biological activity.198 Our study suggests that producing large amountsof human proinsulin in the milk of dairy animals,such as dairy goats and cows, is feasible.

Genetically transformed dairy breeds may beor already have been generated to produce milkwith a modied biochemical composition to meet

specic needs. For example, the transgenic ap-

proach may be used to humanize cow milk byoverexpressing β- and κ-casein variants.197 Themilk casein concentration has already been in-creased to enhance cheese-making efciency.199,200 Human lysozyme is being produced in the milk ofgenetically engineered goats to decrease the rennetclotting time and increase curd strength, leading to





faster cheese making and rmer cheese.201 Increas-ing the lysozyme concentration in goat milk canextend shelf life by causing spoilage bacteria togrow more slowly.201

VIII. CONCLUSION

In the past decade great progress in understandingthe mechanisms of the regulation of milk proteingene expression at multiple levels has been made.Milk protein genes are synergistically regulated byhormones, growth factors, and the ECM, whichactivate specic transcription factors and modi-fy chromatin structure. Alterations in chromatinstructure include post-translational modications

in epigenetic markers and chromatin remodeling,

which both occur at the nucleosome level and ona macroscopic scale. Studies of milk protein generegulation have led to a new eld of biotechnology

in which pharmaceuticals can now be produced inthe milk of transgenic livestock.

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Current Major Advances in the Regulation of Milk Protein Gene Expression

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