Robert N. Eisenman

Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex

Cytosine residues in the sequence 5′CpG (cytosine–guanine) are often postsynthetically methylated in animal genomes. CpG methylation is involved in long-term silencing of certain genes during mammalian development, and in repression of viral genomes,. The methyl-CpG-binding proteins MeCP1 (ref. 5) and MeCP2 (ref. 6) interact specifically with methylated DNA and mediate transcriptional repression. Here we study the mechanism of repression by MeCP2, an abundant nuclear protein that is essential for mouse embryogenesis. MeCP2 binds tightly to chromosomes in a methylation-dependent manner,. It contains a transcriptional-repression domain (TRD) that can function at a distance in vitro and in vivo. We show that a region of MeCP2 that localizes with the TRD associates with a corepressor complex containing the transcriptional repressor mSin3A and histone deacetylases. Transcriptional repression in vivo is relieved by the deacetylase inhibitor trichostatin A, indicating that deacetylation of histones (and/or of other proteins) is an essential component of this repression mechanism. The data suggest that two global mechanisms of gene regulation, DNA methylation and histone deacetylation, can be linked by MeCP2.

Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc

The myc protooncogene family has been implicated in cell proliferation, differentiation, and neoplasia, but its mechanism of function at the molecular level is unknown. The carboxyl terminus of Myc family proteins contains a basic region helix-loop-helix leucine zipper motif (bHLH-Zip), which has DNA-binding activity and has been predicted to mediate protein-protein interactions. The bHLH-Zip region of c-Myc was used to screen a complementary DNA (cDNA) expression library, and a bHLH-Zip protein, termed Max, was identified. Max specifically associated with c-Myc, N-Myc, and L-Myc proteins, but not with a number of other bHLH, bZip, or bHLH-Zip proteins. The interaction between Max and c-Myc was dependent on the integrity of the c-Myc HLH-Zip domain, but not on the basic region or other sequences outside the domain. Furthermore, the Myc-Max complex bound to DNA in a sequence-specific manner under conditions where neither Max nor Myc exhibited appreciable binding. The DNA-binding activity of the complex was dependent on both the dimerization domain and the basic region of c-Myc. These results suggest that Myc family proteins undergo a restricted set of interactions in the cell and may belong to the more general class of eukaryotic DNA-binding transcription factors.

Histone deacetylases associated with the mSin3 corepressor mediate mad transcriptional repression.

Transcriptional repression by Mad-Max heterodimers requires interaction of Mad with the corepressors mSin3A/B. Sin3p, the S. cerevisiae homolog of mSin3, functions in the same pathway as Rpd3p, a protein related to two recently identified mammalian histone deacetylases, HDAC1 and HDAC2. Here, we demonstrate that mSin3A and HDAC1/2 are associated in vivo. HDAC2 binding requires a conserved region of mSin3A capable of mediating transcriptional repression. In addition, Mad1 forms a complex with mSin3 and HDAC2 that contains histone deacetylase activity. Trichostatin A, an inhibitor of histone deacetylases, abolishes Mad repression. We propose that Mad-Max functions by recruiting the mSin3-HDAC corepressor complex that deacetylates nucleosomal histones, producing alterations in chromatin structure that block transcription.

Myc’s broad reach

The role of the myc gene family in the biology of normal and cancer cells has been intensively studied since the early 1980s. myc genes, responding to diverse external and internal signals, express transcription factors (c-, N-, and L-Myc) that heterodimerize with Max, bind DNA, and modulate expression of a specific set of target genes. Over the last few years, expression profiling, genomic binding studies, and genetic analyses in mammals and Drosophila have led to an expanded view of Myc function. This review is focused on two major aspects of Myc: the nature of the genes and pathways that are targeted by Myc, and the role of Myc in stem cell and cancer biology.

Mad: A heterodimeric partner for Max that antagonizes Myc transcriptional activity

Myc family proteins appear to function through heterodimerization with the stable, constitutively expressed bHLH-Zip protein, Max. To determine whether Max mediates the function of regulatory proteins other than Myc, we screened a lambda gt11 expression library with radiolabeled Max protein. One cDNA identified encodes a new member of the bHLH-Zip protein family, Mad. Human Mad protein homodimerizes poorly but binds Max in vitro, forming a sequence-specific DNA binding complex with properties very similar to those of Myc-Max. Both Myc-Max and Mad-Max heterocomplexes are favored over Max homodimers, and, unlike Max homodimers, the DNA binding activity of the heterodimers is unaffected by CKII phosphorylation. Mad does not associate with Myc or with representative bHLH, bZip, or bHLH-Zip proteins. In vivo transactivation assays suggest that Myc-Max and Mad-Max complexes have opposing functions in transcription and that Max plays a central role in this network of transcription factors.

Drosophila myc regulates cellular growth during development.

Transcription factors of the Myc proto-oncogene family promote cell division, but how they do this is poorly understood. Here we address the functions of Drosophila Myc (dMyc) during development. Using mosaic analysis in the fly wing, we show that loss of dMyc retards cellular growth (accumulation of cell mass) and reduces cell size, whereas dMyc overproduction increases growth rates and cell size. dMyc-induced growth promotes G1/S progression but fails to accelerate cell division because G2/M progression is independently controlled by Cdc25/String. We also show that the secreted signal Wingless patterns growth in the wing primordium by modulating dMyc expression. Our results indicate that dMyc links patterning signals to cell division by regulating primary targets involved in cellular growth and metabolism.

Mad-Max transcriptional repression is mediated by ternary complex formation with mammalian homologs of yeast repressor Sin3.

The bHLH-ZIP protein Mad heterodimerizes with Max as a sequence-specific transcriptional repressor. Mad is rapidly induced upon differentiation, and the associated switch from Myc-Max to Mad-Max heterocomplexes seem to repress genes normally activated by Myc-Max. We have identified two related mammalian cDNAs that encode Mad-binding proteins. Both possess sequence homology with the yeast transcription repressor Sin3, including four conserved paired amphipathic helix (PAH) domains. mSin3A and mSin3B bind specifically to Mad and the related protein Mxi1. Mad-Max and mSin3 form ternary complexes in solution that specifically recognize the Mad-Max E box-binding site. Mad-mSin3 association requires PAH2 of mSin3A/mSin3B and the first 25 residues of Mad, which contains a putative amphipathic alpha-helical region. Point mutations in this region eliminate interaction with mSin3 proteins and block Mad transcriptional repression. We suggest that Mad-Max represses transcription by tethering mSin3 to DNA as corepressors and that a transcriptional repression mechanism is conserved from yeast to mammals.

Histone sumoylation is associated with transcriptional repression.

Histone proteins are subject to modifications, such as acetylation, methylation, phosphorylation, ubiquitination, glycosylation, and ADP ribosylation, some of which are known to play important roles in the regulation of chromatin structure and function. Here we report that histone H4 is modified by small ubiquitin-related modifier (SUMO) family proteins both in vivo and in vitro. H4 binds to the SUMO-conjugating enzyme (E2), UBC9, and can be sumoylated in an E1 (SUMO-activating enzyme)- and E2-dependent manner. We present evidence suggesting that histone sumoylation mediates gene silencing through recruitment of histone deacetylase and heterochromatin protein 1.

c-Myc binds to human ribosomal DNA and stimulates transcription of rRNA genes by RNA polymerase I

c-Myc coordinates cell growth and division through a transcriptional programme that involves both RNA polymerase (Pol) II- and Pol III-transcribed genes. Here, we demonstrate that human c-Myc also directly enhances Pol I transcription of ribosomal RNA (rRNA) genes. rRNA synthesis and accumulation occurs rapidly following activation of a conditional MYC-ER allele (coding for a Myc–oestrogen-receptor fusion protein), is resistant to inhibition of Pol II transcription and is markedly reduced by c-MYC RNA interference. Furthermore, by using combined immunofluorescence and rRNA-FISH, we have detected endogenous c-Myc in nucleoli at sites of active ribosomal DNA (rDNA) transcription. Our data also show that c-Myc binds to specific consensus elements located in human rDNA and associates with the Pol I-specific factor SL1. The presence of c-Myc at specific sites on rDNA coincides with the recruitment of SL1 to the rDNA promoter and with increased histone acetylation. We propose that stimulation of rRNA synthesis by c-Myc is a key pathway driving cell growth and tumorigenesis.

Sin meets NuRD and other tails of repression.

Why are there multiple HDAC corepressor complexes? Although the mSin3 and Mi-2/NuRD complexes could be redundant, it is difficult to imagine the high degree of evolutionary conservation of two such multicomponent systems unless some aspects of their function are unique. Indeed both the chromatin remodeling activity associated with Mi-2/NuRD as well as the distinct transcription factors present in both corepressor complexes point toward specialization. Perhaps Mi-2/NuRD target genes are predominantly localized in relatively closed regions of chromatin, thus requiring a nucleosome remodeling step for access of Mi-2/NuRD to histones and subsequent or concomitant deacetylation (see Tyler and Kadonaga 1999xTyler, J.K and Kadonaga, J.T. Cell. 1999; 99: 443–446Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesTyler and Kadonaga 1999). Such genes might be resistant to Sin3-mediated repression, which would be reserved for previously remodeled actively transcribed genes that may not require further chromatin remodeling for deacetylation such as HO in yeast (8xKrebs, J.E, Kuo, M.H, Allis, C.D, and Peterson, C.L. Genes Dev. 1999; 13: 1412–1421Crossref | PubMedSee all References, 3xCosma, M.P, Tanaka, T, and Nasmyth, K. Cell. 1999; 97: 299–311Abstract | Full Text | Full Text PDF | PubMed | Scopus (548)See all References). There is also compelling evidence that certain silenced regions of chromatin may occupy nuclear compartments distinct from those in which active genes reside. Therefore, it is possible that the Mi-2/NuRD (or, for that matter, any of the other corepressors mentioned above) may possess activities that facilitate relocalization of targeted genetic regions to specific nuclear domains (see Sun and Elgin 1999xSun, F.L and Elgin, S.C.R. Cell. 1999; 99: 459–462Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesSun and Elgin 1999 [this issue of Cell]).In a general sense, the mechanism of repression is likely to have gene-specific aspects even though much of the regulatory machinery involved is common to all genes. Different corepressors may be thought of as acting to unleash HDAC activities at the target site in distinct ways. For example, some corepressors may tether HDAC to the complex while others may release HDAC in the vicinity of the gene target. The interplay between different types of chromatin modifications including deacetylation, methylation, and chromatin remodeling may be coordinated through the functions of corepressors (see Bird and Wolffe 1999xBird, A.P and Wolffe, A.P. Cell. 1999; 99: 451–454Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesBird and Wolffe 1999 and Tyler and Kadonaga 1999xTyler, J.K and Kadonaga, J.T. Cell. 1999; 99: 443–446Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesTyler and Kadonaga 1999). Moreover, the recent work on corepressors has illuminated a profound connection between transcriptional repression and fundamental aspects of cell biology including proliferation, differentiation, and cancer.*To whom correspondence should be addressed (e-mail: eisenman@fhcrc.org).