Regulation of E2A Activities by Histone Acetyltransferases in B Lymphocyte Development

Abstract

Genetic studies have demonstrated that the basic helix-loop-helix protein E2A is an essential transcription factor in B lymphocyte lineage commitment and differentiation. However, the mechanism underlying E2A-mediated transcription regulation is not fully understood. Here, we investigated the physical and genetic interactions between E2A and co-activators histone acetyltransferases (HATs) in B cells. Gel filtration analysis of human pre-B cell nuclear extract showed that E2A coelutes with the HATs p300, CBP, and PCAF. A co-immunoprecipitation assay further demonstrated that a fraction of endogenous E2A proteins is associated with each of the three HATs. We show that these HATs acetylate E2A in vitro, enhance E2A-mediated transcription activity, and promote nuclear retention of E2A proteins. A catalytic mutation of p300 completely abrogates the ability of p300 to acetylate E2A and to promote E2A nuclear retention in 293T cells. A breeding test between E2A heterozygous mice and p300 heterozygous mice demonstrated that these two genes interact for proper B cell development. Collectively, these results suggest that E2A and HATs collaboratively regulate B cell development.The development of B lymphocytes in the bone marrow is initiated and tightly regulated by at least three transcription factors, E2A, EBF, and Pax5 (1). Mice missing any one of these transcription factors show complete block in B cell development at the pro-B cell stage (2-5). Although the expression of EBF and Pax5 are relatively restricted to the B cell lineage, E2A is found to be much broadly expressed. Both biochemical and genetic analyses have indicated that E2A is the most upstream regulator among the three transcription factors and is continuously involved in regulating the expression of B cell-specific genes through the later stages of B cell development (6, 7). It is not clear how E2A controls the broad array of tissue-specific and stage-specific gene expression during B cell development.E2A is a founding member of the basic helix-loop-helix (bHLH) 1 transcription factor family, which plays an evolutionarily conserved role in regulating the differentiation events in various tissue types including B lymphocytes in mammals (8).The E2A gene encodes two bHLH transcription factors, E12 and E47, which are generated through differential splicing to two adjacent exons that encode the bHLH domains (9). The bHLH domains are required for protein dimerization and DNA binding (10). Two transactivation domains (AD) are mapped to the amino terminus of the E2A proteins (11, 12). E2A proteins form homodimers or heterodimers through HLH interactions with other broadly expressed bHLH transcription factors such as HEB (13) or with tissue-restricted bHLH transcription factors such as MyoD (14). These bHLH protein dimers bind to DNA at the consensus sequence CANNTG, designated as the E-box. Functional E-box sites are found in the promoters and enhancers of a wide variety of tissue-specific genes including immunoglobulin genes in B cells (15). The ubiquitously exp...

The FHA domain protein SNIP1 is a regulator of the cell cycle and cyclin D1 expression

“…Furthermore, gel filtration analysis using HeLa nuclear extract demonstrated that SNIP1 did not coelute with p300, confirming that these two proteins do not interact in this cell line (Figure 4b). p300 was found to exist in a relatively large complex (B1 MDa) as previously reported (Bradney et al, 2003), but SNIP1 is a component of an even larger, unknown, complex. SNIP1 was not detected in any other fraction of the analysis, suggesting that all cellular SNIP1 is present in this high molecular weight complex (data not shown).…”

Molecular functions of the transcription factors E2A and E2-2 in controlling germinal center B cell and plasma cell development

“…Moreover, few repressed target genes were highly regulated by the two E-proteins in contrast to a large proportion of the activated target genes. In support of transcriptional activation, E-proteins contain 3 activation domains (AD1, AD2, and AD3), which interact with the coactivators p300 and CBP (Bradney et al, 2003;Bayly et al, 2004) and the promoter recognition factor TFI ID (Chen et al, 2013). At the chromatin level, we identified a novel role for E-proteins in shaping the enhancer landscape at its activated target genes, as E2A and E2-2 are responsible for the induction and maintenance of DHS sites containing E2A-binding sites.…”

“…Within the lymphoid system, E-proteins function as homodimers or heterodimers with a different E-protein (Bain et al, 1993;Shen and Kadesch, 1995). E-proteins are thought to mainly function as transcriptional activators, as they interact with the co-activators p300 and CBP (Bradney et al, 2003;Bayly et al, 2004), as well as the promoter recognition factor TFI ID (Chen et al, 2013). The activity of E-proteins is controlled by the inhibitor of DNA binding proteins, which are HLH proteins lacking the basic DNA-binding domain, and are thus capable of sequestering E-proteins into DNA-bindingincompetent heterodimers (Kee, 2009).…”

Notch1 confers thymocytes a resistance to GC-induced apoptosis through Deltex1 by blocking the recruitment of p300 to the SRG3 promoter

“…Recent reports have also shown that coactivator p300, CBP, and PCAF enhance the E2A-mediated transcriptional activity, and promote nuclear retention of E2A proteins in 293T cells. 26 Moreover, p300 can associate with the bHLH region and the amino-terminal region of E2A proteins. 21,25 Furthermore, Deltex1 was reported to bind directly to p300, thereby counteracting p300-dependent transcriptional activation by bHLH protein.…”

“…21 In addition, the interaction mechanism between E2A and p300/ CBP is reported in insulin gene transcription and in pre-B cells, respectively. 26,27 E2A/HEB binding to the E-box element on SRG3 promoter regulates the SRG3 expression and, thereby, controls the sensitivity to GC-induced apoptosis in thymocytes. 18 Therefore, it is possible that Notch-induced Deltex1 may inhibit the transcriptional activity of E2A by blocking the recruitment of a coactivator(s), and this will result in reduced expression of SRG3 and resistance to the GC-induced apoptosis in developing thymocytes.…”