Joan Boyes

Regulation of activity of the transcription factor GATA-1 by acetylation

Modification of histones, DNA-binding proteins found in chromatin, by addition of acetyl groups occurs to a greater degree when the histones are associated with transcriptionally active DNA,. A breakthrough in understanding how this acetylation is mediated was the discovery that various transcriptional co-activator proteins have intrinsic histone acetyltransferase activity (for example, Gcn5p (ref. 3), PCAF, TAFII250 (ref. 5) and p300/CBP,). These acetyltransferases also modify certain transcription factors (TFIIEβ, TFIIF, EKLF and p53 (refs 8–10)). GATA-1 is an important transcription factor in the haematopoietic lineage and is essential for terminal differentiation of erythrocytes and megakaryocytes,. It is associated in vivo with the acetyltransferase p300/CBP. Here we report that GATA-1 is acetylated in vitro by p300. This significantly increases the amount of GATA-1 bound to DNA and alters the mobility of GATA-1–DNA complexes, suggestive of a conformational change in GATA-1. GATA-1 is also acetylated in vivo and acetylation directly stimulates GATA-1-dependent transcription. Mutagenesis of important acetylated residues shows that there is a relationship between the acetylation and in vivo function of GATA-1. Wepropose that acetylation of transcription factors can alter interactions between these factors and DNA and among different transcription factors, and is an integral part of transcription and differentiation processes.

High levels of De Novo methylation and altered chromatin structure at CpG islands in cell lines

CpG islands are normally methylation free in cells of the animal, even when the associated gene is transcriptionally silent. In mouse NIH 3T3 and L cells, however, over half of the islands are heavily methylated. Near identity of the methylated subset in the two cell lines suggested that methylation is confined to genes that are nonessential in culture. In agreement with this, islands at several tissue-specific genes, but not at housekeeping genes, have become methylated in many human and mouse cell lines. At the chromatin level, methylated islands are Mspl resistant compared with their nonmethylated counterparts. We suggest that mutation-like gene inactivation due to CpG island methylation is widespread in many cell lines and could explain the loss of cell type-specific functions in culture.

DNA methylation inhibits transcription indirectly via a methyl-CpG binding protein

We have studied the mechanism by which DNA methylation inhibits transcription both in cell-free nuclear extracts and in the living cell. Repression of transcription in vitro for four different promoters was shown to be an indirect effect. The mediator of repression had properties indistinguishable from those of a methyl-CpG binding protein (MeCP-1) that has been previously identified. Use of differentially methylated promoters and methylated competitors in transient transfection assays suggested that indirect repression via MeCP-1 also occurs in the living cell. This was supported by the fact that MeCP-1-deficient cells showed much reduced repression of methylated genes.

Repression of genes by DNA methylation depends on CpG density and promoter strength: evidence for involvement of a methyl-CpG binding protein.

Repression of transcription from densely methylated genes can be mediated by the methyl‐CpG binding protein MeCP‐1 (Boyes and Bird, 1991). Here we have investigated the effect of methylation on genes with a low density of methyl‐CpG. We found that sparse methylation could repress transfected genes completely, but the inhibition was fully overcome by the presence in cis of an SV40 enhancer. Densely methylated genes, however, could not be reactivated by the enhancer. In vitro studies showed that the sparsely methylated genes bound weakly to MeCP‐1 and that binding interfered with transcription. In the absence of available MeCP‐1, methylation had minimal effects on transcription. From these and other results we propose that sparsely methylated genes form an unstable complex with MeCP‐1 which prevents transcription when the promoter is weak. This complex can be disrupted by a strong promoter, thereby allowing the methylated gene to be transcribed.

Tissue-specific factors additively increase the probability of the all-or-none formation of a hypersensitive site.

DNase I‐hypersensitive sites lack a canonical nucleosome and have binding sites for various transcription factors. To understand how the hypersensitivity is generated and maintained, we studied the chicken erythroid‐specific beta(A)/epsilon globin gene enhancer, a region where both tissue‐specific and ubiquitous transcription factors can bind. Constructions containing mutations of this enhancer were stably introduced into a chicken erythroid cell line. We found that the hypersensitivity was determined primarily by the erythroid factors and that their binding additively increased the accessibility. The fraction of accessible sites in clonal cell lines was quantitated using restriction endonucleases; these data implied that the formation of each hypersensitive site was an all‐or‐none phenomenon. Use of DNase I and micrococcal nuclease probes further indicated that the size of the hypersensitive site was influenced by the binding of transcription factors which then determined the length of the nucleosome‐free gap. Our data are consistent with a model in which hypersensitive sites are generated stochastically: mutations that reduce the number of bound factors reduce the probability that these factors will prevail over a nucleosome; thus, the fraction of sites in the population that are accessible is also diminished.

Regulation of V(D)J recombination by nucleosome positioning at recombination signal sequences

A key component in the regulation of V(D)J recombination is control of the accessibility of RAG proteins to recombination signal sequences (RSS). Nucleosomes are known to inhibit this accessibility. We show here that the signal sequence itself represses accessibility by causing nucleosome positioning over the RSS. This positioning is mediated, in vitro and in vivo, by the conserved nonamer of the RSS. Consistent with this strong positioning, nucleosomes at RSSs are resistant to remodelling by nucleosome sliding. In vivo we find that consensus RSSs are preferentially protected, whereas those that lack a consensus nonamer, including some cryptic RSSs, fail to position nucleosomes. Decreased protection of these non‐consensus RSSs correlates with their increased use in recombination assays. We therefore suggest that nucleosome positioning by RSSs provides a previously unanticipated level of protection and regulation of V(D)J recombination.

Acetylation and MAPK phosphorylation cooperate to regulate the degradation of active GATA-1.

Regulation of transcription requires mechanisms to both activate and terminate transcription factor activity. GATA‐1 is a key haemopoietic transcription factor whose activity is increased by acetylation. We show here that acetylated GATA‐1 is targeted for degradation via the ubiquitin/proteasome pathway. Acetylation positively signals ubiquitination, suggesting that activation by acetylation simultaneously marks GATA‐1 for degradation. Promoter‐specific MAPK phosphorylation then cooperates with acetylation to execute protein loss. The requirement for both modifications is novel and suggests a way by which degradation of the active protein can be specifically regulated in response to external phosphorylation‐mediated signalling. As many transcription factors are activated by acetylation, we suggest that this might be a general mechanism to control transcription factor activity.

Interplay between DNA Methylation and Transcription Factor Availability: Implications for Developmental Activation of the Mouse Myogenin Gene

ABSTRACT During development, gene activation is stringently regulated to restrict expression only to the correct cell type and correct developmental stage. Here, we present mechanistic evidence that suggests DNA methylation contributes to this regulation by suppressing premature gene activation. Using the mouse Myogenin promoter as an example of the weak CpG island class of promoters, we find that it is initially methylated but becomes demethylated as development proceeds. Full hypersensitive site formation of the Myogenin promoter requires both the MEF2 and SIX binding sites, but binding to only one site can trigger the partial chromatin opening of the nonmethylated promoter. DNA methylation markedly decreases hypersensitive site formation that now occurs at a detectable level only when binding to both MEF2 and SIX binding sites is possible. This suggests that the probability of activating the methylated promoter is low until two of the factors are coexpressed within the same cell. Consistent with this, the single-cell analysis of developing somites shows that the coexpression of MEF2A and SIX1, which bind the MEF2 and SIX sites, correlates with the fraction of cells that demethylate the Myogenin promoter. Taken together, these studies imply that DNA methylation helps to prevent inappropriate gene activation until sufficient activating factors are coexpressed.

HSP27 controls GATA-1 protein level during erythroid cell differentiation.

Heat shock protein 27 (HSP27) is a chaperone whose cellular expression increases in response to various stresses and protects the cell either by inhibiting apoptotic cell death or by promoting the ubiquitination and proteasomal degradation of specific proteins. Here, we show that globin transcription factor 1 (GATA-1) is a client protein of HSP27. In 2 models of erythroid differentiation; that is, in the human erythroleukemia cell line, K562 induced to differentiate into erythroid cells on hemin exposure and CD34(+) human cells ex vivo driven to erythroid differentiation in liquid culture, depletion of HSP27 provokes an accumulation of GATA-1 and impairs terminal maturation. More specifically, we demonstrate that, in the late stages of the erythroid differentiation program, HSP27 is phosphorylated in a p38-dependent manner, enters the nucleus, binds to GATA-1, and induces its ubiquitination and proteasomal degradation, provided that the transcription factor is acetylated. We conclude that HSP27 plays a role in the fine-tuning of terminal erythroid differentiation through regulation of GATA-1 content and activity.

Transcription-coupled eviction of histones H2A/H2B governs V(D)J recombination.

Initiation of V(D)J recombination critically relies on the formation of an accessible chromatin structure at recombination signal sequences (RSSs) but how this accessibility is generated is poorly understood. Immunoglobulin light‐chain loci normally undergo recombination in pre‐B cells. We show here that equipping (earlier) pro‐B cells with the increased pre‐B‐cell levels of just one transcription factor, IRF4, triggers the entire cascade of events leading to premature light‐chain recombination. We then used this finding to dissect the critical events that generate RSS accessibility and show that the chromatin modifications previously associated with recombination are insufficient. Instead, we establish that non‐coding transcription triggers IgL RSS accessibility and find that the accessibility is transient. Transcription transiently evicts H2A/H2B dimers, releasing 35–40 bp of nucleosomal DNA, and we demonstrate that H2A/H2B loss can explain the RSS accessibility observed in vivo. We therefore propose that the transcription‐mediated eviction of H2A/H2B dimers is an important mechanism that makes RSSs accessible for the initiation of recombination.