Chromatin structure and the expression of globin-encoding genes

Gene. 135 (1993) 119-124

119

Elsevler Science Publishers B.V. GENE 07425

Chromatin structure and the expression of globin-encoding genes* (Transcription;

trans-acting

factors; GATA-1; locus control region; chromatin domain; nucleosome; supercoiling)

Gary Felsenfeld Laboratory Bethesda,

ofMolecular

Biology, National Institute ofDiabetes

and DIgestwe and Kidney Diseases, Building 5, Room 212, National Institutes of Health,

MD 20892, USA

Receivedby G. BernardI: 7 June 1993;Accepted: 11 June 1993;Receivedat publishers: 16 July 1993

SUMMARY

The developmental regulation of globin gene expression in the chicken has been studied. All of the genes are regulated by a small number of general erythroid factors. In addition, expression of individual members of the family must be controlled in a lineage (stage)-specific manner. In some cases, the relevant factors may be stage specific, but in others they are not confined to one stage, but exert their control through developmentally regulated changes in their abundance within the nucleus. Chromatin structural elements, such as locus control regions and insulators, are also involved in control of eukaryotic gene expression. Because so much is understood about regulation of individual genes, the globin family has proven valuable in investigating control of transcription at the level of chromatin structure.

attempting to define the cis-regulatory elements and trans-acting factors that control expression of these genes.

INTRODUCTION Regulation mately

of gene expression

be understood

in eukaryotes

in the context

must ulti-

of the chromatin

To address questions about the relationship between chromatin structure and gene expression, it is useful to choose a system of genes that is modulated in expression during development. The family of avian globin genes provides such a system. We have used the CI-and /?-globin genes of the chicken to study the changes in chromatin that accompany gene activation. Of necessity we began by structure

in which the genes are embedded.

Correspondence

to: Dr. G. Felsenfeld, Laboratory

National

Institute

of Diabetes

Building

5, Room

212, National

and

Digestive

Institutes

of Molecular and

Kidney

of Health,

Biology, Diseases,

Bethesda,

20892. USA. Tel. (l-301) 496-4173; Fax (l-301) 496-0201. *Presented at the COGENE Symposium, ‘From the Double the Human Genome: 21-23 April 1993.

40 Years of Molecular

Genetics’, UNESCO,

MD

Helix to Pans,

Abbrewations. bp, base pair(s); CAT, chloramphenicol acetyltransferase: cat, gene encoding CAT; Eryfl, transcription factor; GATA-1, transcription factor; kb, kilobase or 1000 bp; LCR, locus control region; NF-1, transcription NF-E2 transcrlption factor; factor: PAL, transcrlption factor; Spl, transcription factor.

GLOBIN

GENE

EXPRESSION

In the developing chick embryo (Fig. l), the primitive lineage cells appearing in the circulation up to day 4-5 after fertilization express the embryonic P-globin genes p and E, the embryonic a gene, 71(or c?), and two other M genes, tl* and aD. Beginning at day 4-5, the definitive lineage appears, expressing the adult genes, DA and fin, as well as aA and CI~. Two questions raised by this sequence of events are: what turns on the entire family of globin genes in cells of the erythroid lineage, and what turns individual members of the family on and off in a stage-specific manner? Connections between chromatin structure and transcriptional activation have been evident since the early work of Weintraub and his collaborators (Weintraub and Groudine, 1976), which established that the chromatin of active genes is somewhat more sensitive to attack by DNase I than that of inactive genes in the same cell. In

120 Days of Development

1

5-

P

10 -15

e;a

c

Cluster

PH PA

P

e

E

HbA = ~$32”

a

HbO=cfq

Primitiveb@

drT$

Cluster

HbH = $B2”

a Fig 2. Arrangement symbol Fig 1. Erythrold embryomc

cell lineages

clrculatlon

durmg

in the chlcken development.

species, with their component

globm

P. E. M, A. D, and H are abbreviated nent globm

chams.

coded

as they appear

The tetramerlc

chains,

are shown.

m the

hemoglobin Hemoglobins

by the individual

corresponding

genes, are

of Dr Mark

Mmle. data

the chicken /?-globin domain, this ‘general’ nuclease sensitivity extends for many kb on either side of the gene cluster. A second kind of perturbation in chromatin structure, site, is more limited

in extent

and is associated with promoters, enhancers, and other binding sites for regulatory factors (Wu et al., 1979; Groudine and Weintraub, 1981). Our earliest studies of the chicken PA-globin gene accordingly focused on the hypersensitive site at the promoter ( McGhee et al., 1981). and on the mechanism of generation of hypersensitivity. We carried out chromatin reconstitution experiments with core histones and plasmids carrying the promoter, and found that in the presence of erythroid nuclear extracts it is possible to regenerate a hypersensitive site (Emerson and Felsenfeld, The search in erythroid for this behavior quickly

the posltlon and

/P’-glob

r-cluster

enhancer.

aD

aA

t

of the r- and /,‘-&&vI genes m the chicken. t genes.

of the p-cluster The symbol

All other symbols

enhancer.

I marks

located

the positton

mark the poaltlons

The

between of the

of the mdlvl-

dual members of the globm gene family. ah shown m the legend to Fig

1

as HbP. HbE. etc. Theu compo-

shown as Y. xA. c?‘. E, p. p”. and p” (courtesy from Bruns and Ingram. 1973)

the nuclease-hypersensitive

the

Y marks

IT

1984 ). extracts for factors responsible led to the realization that the

assay for hypersensitivity was too cumbersome, and that more direct methods for studying protein-DNA interaction were required. DNase I footprinting and gel assays provided us with detailed information about binding (Emerson et al., 1985). We were also aided by the development of a method for introducing plasmids directly into primary erythroid cells, which made it possible to study expression at each stage of erythroid development (Hesse et al., 1986). Application of this method revealed the presence downstream from the /I” gene and upstream from e-globin of a strong, erythroid-specific enhancer (see Fig. 2) which functioned at all stages of erythroid development (Choi and Engel, 1986; Hesse et al., 1986). The enhancer contained binding sites for a novel factor (Evans et al., 1988: Reitman and Felsenfeld. 1988; Wall et al., 1988; Evans and Felsenfeld, 1989; Tsai et al., 1989:

Trainor et al., 1990; Zon et al., 1990; Hannon et al.. 1991 ). which we named Eryf 1 (now called GATA- 1 1. GATA-1 (as well as NF-E2, a member of the AP-I family that is expressed in erythroid cells: Andrews et al.. 1993) appears to be an important determinant of erythroid-specific expression. Binding sites for GATA- I were found near all of the chicken globin genes (Evans et al., 1988), the genes of the human P-globin cluster, and other erythroid-specific genes. The expression of GATA-1 is confined to erythroid lineage cells, mast cells, and megakaryocytes (Martin et al.. 1990). and is also found in testes (Ito et al., 1993). GATA-1 is essential: knocking out the gene in transgenic mice is lethal (Pevny et al., 1991). It

seems likely that the establishment and maintenance of erythroid-specific gene expression depends upon the action of a small number of factors including GATA-1 and NF-E2. A variety of mechanisms are involved in the control of lineage-specific (‘stage’-specific) expression of the individual

globin

genes. In the case of chicken

stage-specific factor, NF-EB has been important role (Choi and Engel, 1988) pression in definitive lineage cells. A mechanism appears to be involved

DA-globin,

a

shown to play an in turning on exdifferent kind of in regulation of

p-globin (Minie et al., 1992). an embryonic P-globin, and x-globin (Knezetic and Felsenfeld. 1993 ). an embryonic a-globin. In the case of the n gene, plasmids carrying about 350 bp of the upstream region containing the promoter, coupled to the cat reporter gene, were transiently transfected into primary erythroid cells. The patterns of expression from promoters of the a-gene family are shown in Fig. 3. About the same levels of expression of X’ and X” were seen m primitive and definitive lineages, but the 71 promoter shows very strong dependence of expression on developmental stage. This promoter is nearly inactive in definitive lineage cells. Thus, the promoters of all three x genes contain information for stage-

121

. ..

Definltlve

Spl

Y-Box

NF-1

site

site

Spl site

Y

NF-1

q

A

+++

m __~._

Fig. 4 Model chicken

abundance P:

P:J

P:RSV

PLASMID

D:J

CONSTFWCTION

regions

5’ of the regton

shows that the gene is transcripttonally

J (c( cluster enhancer. enhancer)

marked

(from Knezetic

specific expression

i in Fig. 2) or RSV (Rous

and Felsenfeld.

sarcoma

and Felsenfeld,

of the

and the relative

development. Three GATA-1 sites regulation of the CL”promoter are

further

Knezettc

with

regulation

shows the most proxi-

(Spl. Y-box, and NF-1) that

arrow

cells but this transcrtptton

coupled

proteins

found

mg: P (tr promoter),

A (a* promoter),

Site

of the cc”-globrn gene promoter

of the three regulatory

Fig. 3. Expression of the cat reporter gene m primary primitive or definitive lineage cells after transient transfection with plasmids containD (aD promoter).

Sib

v+

of developmental

bind to these regions throughout also involved in the transcriptional

A:J

NF-1

cc”-globin gene The figure schematically

mal regulatory 0

of the mechanism

y-&x

dtagrammed.

is blocked

The rightward

pointing

active m the prtmtttve

m the definitive

cell types (from

1993).

virus

1993).

that mimics the program

observed

in

vivo (see Fig. 1). Similar behavior is seen with the p-g&in gene promoter (Minie et al., 1992). The elements in the TCpromoter responsible for this behavior have been identified by footprinting and gel mobility assays with nuclear extracts, coupled with transient transfection assays with mutated DNA sequences. Because multiple interactions are involved, mutations at the various binding sites must be carried out in all possible combinations. The results implicate three factors in

tory element. GATA-1 also decreases in abundance. overall effect is to suppress expression. Expression

The from

the p gene promoter is also controlled by concentration effects (Minie et al., 1992), but in that case Spl and GATA-1 play the important roles. Although some caution must be used in extrapolating results obtained with truncated promoters in transient expression assays, it seems likely that mechanisms of this kind play a role in vivo in the stage-specific expression of rc and p.

this behavior: PAL (a member of the NF-1 family of regulatory factors), an Spl-like protein, and a Y-box protein

CHROMATIN

that binds to an extended CCAAT sequence motif (Knezetic and Felsenfeld, 1993). In addition, there are

(a) Locus control regions

three GATA-1 sites further upstream. The mechanism giving rise to lineage-specific expression is not obvious, since all of these are present in both primitive and defin-

events at local promoters and enhancers and the structure of the chromatin that surrounds the genes, and in which they are packaged? We still do not understand the genesis of hypersensitive sites; it is possible that more than one mechanism is involved. In some cases, hypersensitivity can be generated in a very short period of time in non-

itive lineages. However, in the course of work on the p gene (Minie et al., 1992) it was demonstrated that while PAL remains fairly constant in abundance, there is fiveto tenfold less Spl and GATA-1 in definitive cell nuclei than in the nuclei of primitive cells. The Y-box protein undergoes a similar diminution (Knezetic and Felsenfeld, 1993). The lineage-dependent behavior observed for the 7c constructs can be explained by this change (Fig. 4). In primitive cells, the abundance of Spl and Y-box protein is high: Y and NF-1 sites overlap, and Y competitively displaces NF-1; the promoter is active. In definitive cells Y and Spl site occupancy has dropped. NF-1, which remains at constant concentration, can compete for the Y site. Furthermore, NF-1 acts on Spl as a negative regula-

What

STRUCTURE

is the

relationship

between

these

regulatory

dividing cells by the binding of factors which are capable of recognizing sites that are in the midst of nucleosomecovered regions (Bresnick et al., 1992). This effect can be duplicated in vitro under some circumstances, e.g., through the use of artificial promoters with multiple binding sites for factors such as GAL4-VP16 (Workman and Kingston, 1992). Under other circumstances, it has proven difficult to displace or disrupt promoter-bound nucleosome core particles by the addition of trans-acting factors (Laybourn and Kadonaga, 1991). Such results give rise to the suggestion that some hypersensitive sites

122 are generated

during

replication,

when there is a tempo-

rary decrease in the density of histones, and trtrns-acting factors can bind pre-emptively (Felsenfeld, 1992). Other ment.

hypersensitive

first

identified

1985).

Studies

deletion

Experiments

was

(LXR). These

of human

P-thalassemias

essential

in transgenic

were coupled

mice showed

to globin

dent on the number

Isolated

produce

equivalent

supe~helical

located

(Tuan

et al.,

arising

from

of the

genes.

was linearly

of boundary elements. We may think of LCRs

unconstrained

( positive)

supercoiled

in front of it, and negative

et al.,

{P’

every in an

that

f Liu and Wang, might

spontaneously

possible

that

density.

an advancing

transielltl~/

1987 ). suggested

be displaced

It even seemed

forming

a

DNA should

superhelical

complex

can

is to

since such an event raises the

supercoils

that might be expected elements

disfavored,

a histonc

stress in the re-

reason,

scription

with the chicken

as ‘dominant’

core on positively

be energetically

this

of the

the region containing the /I-plohin cluster. However, we have recently obtained evidence (Chung et al.. 1993) that other elements in the 5’ region of both the chicken and have properties

For

of the

amount proportional to the number of integrated copies. The fact that this LCR element is located not distant from the cluster. but within it, makes it clear that the globin LCRs do not necessarily mark the ends of a large chromatin loop domain that might serve to demarcate

p clusters

of the segment.

nucleosome total

positive

of one

DNA segment

that under some conditions

depen-

(Grosveld

mainder

turn, the effect of binding

discovery

that when LCR

promoter and gene in their natural configuration, mouse carrying the gene expresses PA-globin

human

by the generation

to a topologically

pendence of expression. An element with a similar function has been identified within the chicken ~-g~u~j~z locus: it is the PA!&enhancer described earlier. When it is intromice together

is accompanied

\tructurc

of a nucleosome

superhelical

1987; Talbot et al., 1989). In contrast, mice carrying the gene without the LCR did not show such position inde-

duced into transgenic

core particle

ohroniatin

a It ‘eel

negative

of the site of integration

of copies integrated

\vould

Since the formation

octamer

genes, expression

and the level of expression

densit!

the nucleus.

sites were

et al., 1983) revealed that

to expression

within

kind of ele-

~-~~(~~~lr locus,

from the gene cluster

genes was independent construct,

human

of this region (Kioussis

DNA

elements

region

in the

10-20 kb upstream

this

sites mark a different

the Iocus control

superhells

generate

during

normal

positive

supcrcoils

that histone

The tran-

behind octamers

transcription.

nucleosome

core

particles could not be formed on positively supercolled DNA. To test this hypothesis. we attempted to reconstitute mInIchromosomes from histone octamers and positively supercoiled plasmids. The resulting complexes

were examined

by a variety

of physical methods. includmg histone cross-linking and circular dichroism (Clark and Felsenfeld, 199 1). More recently, the hydrodynamic behavior of the complexes has been studied in the analytical ultracentrifuge (D. Clark, R. Ghirlando, G.F. and H. Eisenberg. unpLlblished data) In every case, the complex with positively supercoiled DNA behaves as would be expected if normally folded nucleosome Although

core particles were present. moderate positive supercoiling

does

not

appear to be sufficient to disrupt nucleosomes. thermodynamic conslderatlons guarantee that under reversible conditions, histone octamers will choose negatively supercoiled

regions in preference

to positive.

Direct compe-

help to activate nearby promoters even when the genes are embedded in an otherwise inactive chromatin environment. It is not clear whether this activation involves direct contact between LCR and promoter. There is, however. reason to think that chromatin structure plays a

tition experiments confirm this expectation. and have led us to suggest (Clark and Felsenfeld, 1991) that the transient generation of positive supercolling ahead of a transcription complex, and negative behind, might provide a driving force for the sequential transfer of histones out

role, since at least in some cases LCR elements have a stimulatory effect on transcription only when integrated

of the path of the advancing polymerase (Fig. S ). It is not yet clear whether there 1s suflicient supercolling to drive such a reaction, or whether it actually occurs. Although subsequent studies have led to a detailed description of the fate of nucleosomes on transcription (Clark and Felsenfeld, 1992). it 1s not easy to devise experiments that directly test the role of transient supercoiling in the process of octamer transfer. In trying to assess the contribution of supercoiling to changes m chromatin structure affecting gene activation or repression, it is necessary to take into account not only the forces giving rise to supercoiling, but also the extent of the isolated topological domain over which these forces exert their effect. If a moderate change in supercoiling is dissipated over a large

into the genome, and not in transient transfection experiments (see Felsenfeld, 1992). To understand how LCRs and other chromatin-related functional elements work, it will clearly be necessary to examine their effects on chromatin structure as well as on transcriptional activation (see Reitman et al. ( 1993). for experiments that begin to address such questions). (b) DNA supercoiling and cbromatin structure There is considerable evidence for the involvement of DNA supercoiling in transcriptional control mechanisms, and it has often been suggested that local changes in

123 Chow, O.-R. and Engel, J.D.: A 3’ enhancer

Positive Supercoil

tissue-specific

transcriptional

B-globin gene. Nature

is required

activatton

for temporal

the

and

chicken

adult

323 (1986) 731-734.

Choi, O.-R. and Engel, J.D.: Developmental switching.

of

regulation

of B-globin gene

Cell 55 (1988) 17-26.

Chung, J.H.. Whiteley, p-globm

domain

protects

against

M. and Felsenfeld, G.: A 5’ element of the chicken

serves as an insulator position-effect

m human

in

erythrotd

Drosophila.

Cell

cells and 74 (1993)

505-514. Clark.

e-11111 Fig 5. A model proposed

-

Negative Supercoil

to explain how generation

tive supercoihng ahead of a transcription percoiling behind, could facilitate the polymerase

through

N = nucleosome

D. and Felsenfeld.

supercoiled

histone-covered

(from Clark

DNA.

and Felsenfeld,

of transient

Clark. posi-

complex, and negative supassage of an advancing POL = RNA polymerase: 1991)

D. and Felsenfeld,

Emerson,

G.: A nucleosome

B.M., Lewis, C.D factors adult

with

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conferring

nuclease

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out of

( 1992) 11-22.

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region

of the

domain.

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Position-independent,

Investigations of the globin genes of human, chicken, mouse and other animals have provided a great deal of information about developmental regulation of gene expression. Because they have become model systems for such studies, these genes are also the obvious choice in attempts to go beyond issues of local control of expression to study the contribution of chromatin structure to regulatory processes. As our understanding of the role of chromatin structural elements such as locus control regions and insulating elements increases, we may also have the opportunity to construct more informative experimental systems in vitro. We can hope ultimately to combine cell biology and physical chemistry in a successful attack on these problems.

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core is transferred Cell 71

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CONCLUSIONS

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B.M. and Felsenfeld,

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of nucleosomes

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the path of a transcribing

nuclear

chromatin loop domain, it is unlikely to be important. If on the other hand the same change is topologically confined to a small region, large local superhelix densities can be generated. Slow relaxation of transiently generated supercoiling could have this effect, as could structures in which DNA between adjacent nucleosomes, or even within one, is tethered (V. Studitsky, D. Clark, G.F., unpublished data).

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