Study of the retinoic acid regulated genes in the rodent hypothalamus controlling growth and appetite and the potential for the epigenetic control

Dissertação de mestrado em Bioquímica (área de especialização em Biomedicina)

E2F activation of S phase promoters via association with HCF-1 and the MLL family of histone H3K4 methyltransferases.

E2F transcriptional regulators control human-cell proliferation by repressing and activating the transcription of genes required for cell-cycle progression, particularly the S phase. E2F proteins repress transcription in association with retinoblastoma pocket proteins, but less is known about how they activate transcription. Here, we show that the human G1 phase regulator HCF-1 associates with both activator (E2F1 and E2F3a) and repressor (E2F4) E2F proteins, properties that are conserved in insect cells. Human HCF-1-E2F interactions are versatile: their associations and binding to E2F-responsive promoters are cell-cycle selective, and HCF-1 displays coactivator properties when bound to the E2F1 activator and corepressor properties when bound to the E2F4 repressor. During the G1-to-S phase transition, HCF-1 recruits the mixed-lineage leukemia (MLL) and Set-1 histone H3 lysine 4 methyltransferases to E2F-responsive promoters and induces histone methylation and transcriptional activation. These results suggest that HCF-1 induces cell-cycle-specific transcriptional activation by E2F proteins to promote cell proliferation.

A proline-rich TGF-beta-responsive transcriptional activator interacts with histone H3.

The molecular mechanisms involved in the regulation of gene expression by transforming growth factor-beta (TGF-beta) have been analyzed. We show that TGF-beta specifically induces the activity of the proline-rich trans-activation domain of CTF-1, a member of the CTF/NF-I family of transcription factors. A TGF-beta-responsive domain (TRD) in the proline-rich transcriptional activation sequence of CTF-1 was shown to mediate TGF-beta induction in NIH-3T3 cells. Mutagenesis studies indicated that this domain is not the primary target of regulatory phosphorylations, suggesting that the growth factor may regulate a CTF-1-interacting protein. A two-hybrid screening assay identified a nucleosome component, histone H3, as a specific CTF-1-interacting protein in yeast. Furthermore, the CTF-1 trans-activation domain was shown to interact with histone H3 in both transiently and stably transfected mammalian cells. This interaction requires the TRD, and it appears to be upregulated by TGF-beta in vivo. Moreover, point mutations in the TRD that inhibit TGF-beta induction also reduce interaction with histone H3. In vitro, the trans-activation domain of CTF-1 specifically contacts histone H3 and oligomers of histones H3 and H4, and full-length CTF-1 was shown to alter the interaction of reconstituted nucleosomal cores with DNA. Thus, the growth factor-regulated trans-activation domain of CTF-1 can interact with chromatin components through histone H3. These findings suggest that such interactions may regulate chromatin dynamics in response to growth factor signaling.

Cancer genes hypermethylated in human embryonic stem cells.

Developmental genes are silenced in embryonic stem cells by a bivalent histone-based chromatin mark. It has been proposed that this mark also confers a predisposition to aberrant DNA promoter hypermethylation of tumor suppressor genes (TSGs) in cancer. We report here that silencing of a significant proportion of these TSGs in human embryonic and adult stem cells is associated with promoter DNA hypermethylation. Our results indicate a role for DNA methylation in the control of gene expression in human stem cells and suggest that, for genes repressed by promoter hypermethylation in stem cells in vivo, the aberrant process in cancer could be understood as a defect in establishing an unmethylated promoter during differentiation, rather than as an anomalous process of de novo hypermethylation.

Chromatin domain boundaries delimited by a histone-binding protein in yeast.

When located next to chromosomal elements such as telomeres, genes can be subjected to epigenetic silencing. In yeast, this is mediated by the propagation of the SIR proteins from telomeres toward more centromeric regions. Particular transcription factors can protect downstream genes from silencing when tethered between the gene and the telomere, and they may thus act as chromatin domain boundaries. Here we have studied one such transcription factor, CTF-1, that binds directly histone H3. A deletion mutagenesis localized the barrier activity to the CTF-1 histone-binding domain. A saturating point mutagenesis of this domain identified several amino acid substitutions that similarly inhibited the boundary and histone binding activities. Chromatin immunoprecipitation experiments indicated that the barrier protein efficiently prevents the spreading of SIR proteins, and that it separates domains of hypoacetylated and hyperacetylated histones. Together, these results suggest a mechanism by which proteins such as CTF-1 may interact directly with histone H3 to prevent the propagation of a silent chromatin structure, thereby defining boundaries of permissive and silent chromatin domains.

Sense and antisense transcripts in the histone H1 (HIS-1) locus of Leishmania major.

Histone H1 in the parasitic protozoan Leishmania is a developmentally regulated protein encoded by two genes, HIS-1.1 and HIS-1.2. These genes are separated by approximately 20 kb of sequence and are located on the same DNA strand of chromosome 27. When Northern blots of parasite RNA were probed with HIS-1 strand-specific riboprobes, we detected sense and antisense transcripts that were polyadenylated and developmentally regulated. When the HIS-1.2 coding region was replaced with the coding region of the neomycin phosphotransferase gene, antisense transcription of this gene was unaffected, indicating that the regulatory elements controlling antisense transcription were located outside of the HIS-1.2 gene, and that transcription in Leishmania can occur from both DNA strands even in the presence of transcription of a selectable marker in the complementary strand. A search for other antisense transcripts within the HIS-1 locus identified an additional transcript (SC-1) within the intervening HIS-1 sequence, downstream of adenine and thymine-rich sequences. These results show that gene expression in Leishmania is not only regulated polycistronically from the sense strand of genomic DNA, but that the complementary strand of DNA also contains sequences that could drive expression of open reading frames from the antisense strand of DNA. These findings suggest that the parasite has evolved in such a way as to maximise the transcription of its genome, a mechanism that might be important for it to maintain virulence.

Lausannevirus, a giant amoebal virus encoding histone doublets.

Large viruses infecting algae or amoebae belong to the NucleoCytoplasmic Large DNA Viruses (NCLDV) and present genotypic and phenotypic characteristics that have raised major interest among microbiologists. Here, we describe a new large virus discovered in Acanthamoeba castellanii co-culture of an environmental sample. The virus, referred to as Lausannevirus, has a very limited host range, infecting Acanthamoeba spp. but being unable to infect other amoebae and mammalian cell lines tested. Within A. castellanii, this icosahedral virus of about 200 nm exhibits a development cycle similar to Mimivirus, with an eclipse phase 2 h post infection and a logarithmic growth leading to amoebal lysis in less than 24 h. The 346 kb Lausannevirus genome presents similarities with the recently described Marseillevirus, sharing 89% of genes, and thus belongs to the same family as confirmed by core gene phylogeny. Interestingly, Lausannevirus and Marseillevirus genomes both encode three proteins with predicted histone folds, including two histone doublets, that present similarities to eukaryotic and archaeal histones. The discovery of Lausannevirus and the analysis of its genome provide some insight in the evolution of these large amoebae-infecting viruses.

Transcription factor CTF1 acts as a chromatin domain boundary that shields human telomeric genes from silencing.

Telomeres are associated with chromatin-mediated silencing of genes in their vicinity. However, how epigenetic markers mediate mammalian telomeric silencing and whether specific proteins may counteract this effect are not known. We evaluated the ability of CTF1, a DNA- and histone-binding transcription factor, to prevent transgene silencing at human telomeres. CTF1 was found to protect a gene from silencing when its DNA-binding sites were interposed between the gene and the telomeric extremity, while it did not affect a gene adjacent to the telomere. Protein fusions containing the CTF1 histone-binding domain displayed similar activities, while mutants impaired in their ability to interact with the histone did not. Chromatin immunoprecipitation indicated the propagation of a hypoacetylated histone structure to various extents depending on the telomere. The CTF1 fusion protein was found to recruit the H2A.Z histone variant at the telomeric locus and to restore high histone acetylation levels to the insulated telomeric transgene. Histone lysine trimethylations were also increased on the insulated transgene, indicating that these modifications may mediate expression rather than silencing at human telomeres. Overall, these results indicate that transcription factors can act to delimit chromatin domain boundaries at mammalian telomeres, thereby blocking the propagation of a silent chromatin structure.

Histone deacetylase inhibitors impair innate immune responses to Toll-like receptor agonists and to infection.

Regulated by histone acetyltransferases and deacetylases (HDACs), histone acetylation is a key epigenetic mechanism controlling chromatin structure, DNA accessibility, and gene expression. HDAC inhibitors induce growth arrest, differentiation, and apoptosis of tumor cells and are used as anticancer agents. Here we describe the effects of HDAC inhibitors on microbial sensing by macrophages and dendritic cells in vitro and host defenses against infection in vivo. HDAC inhibitors down-regulated the expression of numerous host defense genes, including pattern recognition receptors, kinases, transcription regulators, cytokines, chemokines, growth factors, and costimulatory molecules as assessed by genome-wide microarray analyses or innate immune responses of macrophages and dendritic cells stimulated with Toll-like receptor agonists. HDAC inhibitors induced the expression of Mi-2β and enhanced the DNA-binding activity of the Mi-2/NuRD complex that acts as a transcriptional repressor of macrophage cytokine production. In vivo, HDAC inhibitors increased the susceptibility to bacterial and fungal infections but conferred protection against toxic and septic shock. Thus, these data identify an essential role for HDAC inhibitors in the regulation of the expression of innate immune genes and host defenses against microbial pathogens.

dKDM5/LID regulates H3K4me3 dynamics at the transcription-start site (TSS) of actively transcribed developmental genes

H3K4me3 is a histone modification that accumulates at the transcription-start site (TSS) of active genes and is known to be important for transcription activation. The way in which H3K4me3 is regulated at TSS and the actual molecular basis of its contribution to transcription remain largely unanswered. To address these questions, we have analyzed the contribution of dKDM5/LID, the main H3K4me3 demethylase in Drosophila, to the regulation of the pattern of H3K4me3. ChIP-seq results show that, at developmental genes, dKDM5/LID localizes at TSS and regulates H3K4me3. dKDM5/LID target genes are highly transcribed and enriched in active RNApol II and H3K36me3, suggesting a positive contribution to transcription. Expression-profiling show that, though weakly, dKDM5/LID target genes are significantly downregulated upon dKDM5/LID depletion. Furthermore, dKDM5/LID depletion results in decreased RNApol II occupancy, particularly by the promoter-proximal Pol lloser5 form. Our results also show that ASH2, an evolutionarily conserved factor that locates at TSS and is required for H3K4me3, binds and positively regulates dKDM5/LID target genes. However, dKDM5/LID and ASH2 do not bind simultaneously and recognize different chromatin states, enriched in H3K4me3 and not, respectively. These results indicate that, at developmental genes, dKDM5/LID and ASH2 coordinately regulate H3K4me3 at TSS and that this dynamic regulation contributes to transcription.

Leishmania major: histone H1 gene expression from the sw3 locus.

Histone H1 in the parasitic protozoan Leishmania is a developmentally regulated protein encoded by the sw3 gene. Here we report that histone H1 variants exist in different Leishmania species and strains of L. major and that they are encoded by polymorphic genes. Amplification of the sw3 gene from the genome of three strains of L. major gave rise to different products in each strain, suggesting the presence of a multicopy gene family. In L. major, these genes were all restricted to a 50-kb Bg/II fragment found on a chromosomal band of 1.3 Mb (chromosome 27). The detection of RFLPs in this locus demonstrated its heterogeneity within several species and strains of Leishmania. Two different copies of sw3 (sw3.0 and sw3.1) were identified after screening a cosmid library containing L. major strain Friedlin genomic DNA. They were identical in their 5' UTRs and open reading frames, but differed in their 3' UTRs. With respect to the originally cloned copy of sw3 from L. major strain LV39, their open reading frames lacked a repeat unit of 9 amino acids. Immunoblots of L. guyanensis parasites transfected with these cosmids revealed that both copies could give rise to the histone H1 protein. The characterization of this locus will now make possible a detailed analysis of the function of histone H1 in Leishmania, as well as permit the dissection of the molecular mechanisms governing the developmental regulation of the sw3 gene.

dKDM5/LID regulates H3K4me3 dynamics at the transcription-start site (TSS) of actively transcribed developmental genes

H3K4me3 is a histone modification that accumulates at the transcription-start site (TSS) of active genes and is known to be important for transcription activation. The way in which H3K4me3 is regulated at TSS and the actual molecular basis of its contribution to transcription remain largely unanswered. To address these questions, we have analyzed the contribution of dKDM5/LID, the main H3K4me3 demethylase in Drosophila, to the regulation of the pattern of H3K4me3. ChIP-seq results show that, at developmental genes, dKDM5/LID localizes at TSS and regulates H3K4me3. dKDM5/LID target genes are highly transcribed and enriched in active RNApol II and H3K36me3, suggesting a positive contribution to transcription. Expression-profiling show that, though weakly, dKDM5/LID target genes are significantly downregulated upon dKDM5/LID depletion. Furthermore, dKDM5/LID depletion results in decreased RNApol II occupancy, particularly by the promoter-proximal Pol lloser5 form. Our results also show that ASH2, an evolutionarily conserved factor that locates at TSS and is required for H3K4me3, binds and positively regulates dKDM5/LID target genes. However, dKDM5/LID and ASH2 do not bind simultaneously and recognize different chromatin states, enriched in H3K4me3 and not, respectively. These results indicate that, at developmental genes, dKDM5/LID and ASH2 coordinately regulate H3K4me3 at TSS and that this dynamic regulation contributes to transcription.

Histone H1 expression varies during the Leishmania major life cycle.

The deduced amino acid sequence of Leishmania major sw3 cDNA reveals the presence of characteristic histone H1 amino acid motifs. However, the open reading frame is of an unusually small size for histone H1 (105 amino acids) because it lacks the coding potential for the central hydrophobic globular domain of linker histones present in other eukaryotes. Here, we provide biochemical evidence that the SW3 protein is indeed a L. major nuclear histone H1, and that it is differentially expressed during the life cycle of the parasite. Due to its high lysine content, the SW3 protein can be purified to a high degree from L. major nuclear lysates with 5% perchloric acid, a histone H1 preparative method. Using an anti-SW3 antibody, this protein is detected as a 17 kDa or as a 17/19 kDa doublet in the nuclear subfraction in different L. major strains. The nuclear localization of the SW3 protein is further supported by immunofluorescence studies. During in vitro promastigote growth, both the sw3 cytoplasmic mRNA and its protein progressively accumulate within parasites from early log phase to stationary phase. Within amastigotes, the high level of H1 expression is maintained but decreases when amastigotes differentiate into promastigotes. Together, these observations suggest that the different levels of this histone H1 protein could influence the varying degrees of chromatin condensation during the life-cycle of the parasite, and provide us with tools to study this mechanism.

Ash2 acts as an Ecdysone Receptor coactivator by stabilizing the histone methyltransferase Trr.

The molting hormone ecdysone triggers chromatin changes via histone modifica- tions that are important for gene regulation. On hormone activation, the ecdysone receptor (EcR) binds to the SET domain-containing histone H3 methyltransferase trithorax-related protein (Trr). Methylation of histone H3 at lysine 4 (H3K4me), which is associated with tran- scriptional activation, requires several cofactors, including Ash2. We find that ash2 mutants have severe defects in pupariation and metamorphosis due to a lack of activation of ecdy- sone-responsive genes. This transcriptional defect is caused by the absence of the H3K4me3 marks set by Trr in these genes. We present evidence that Ash2 interacts with Trr and is re- quired for its stabilization. Thus we propose that Ash2 functions together with Trr as an ecdysone receptor coactivator.

A role for histone-like protein H1 (H-NS) in the regulation of hemolysin expression by Serratia marcescens

The histone-like protein H1 (H-NS) is an abundant structural component of the bacterial nucleoid and influences many cellular processes including recombination, transcription and transposition. Mutations in the hns gene encoding H-NS are highly pleiotropic, affecting the expression of many unrelated genes. We have studied the role of H-NS on the regulation of hemolysin gene expression in Serratia marcescens. The Escherichia coli hns mutant carrying S. marcescens hemolysin genes on a plasmid constructed by ligation of the 3.2-kb HindIII-SacI fragment of pR02 into pBluescriptIIKS, showed a high level of expression of this hemolytic factor. To determine the osmoregulation of wild-type and hns defective mutants the cells were grown to mid-logarithmic phase in LB medium with 0.06 or 0.3 M NaCl containing ampicillin and kanamycin, whereas to analyze the effect of pH on hemolysin expression, the cells were grown to late-logarithmic phase in LB medium buffered with 0.1 M Tris-HCl, pH 4.5 to 8.0. To assay growth phase-related hemolysin production, bacterial cells were grown in LB medium supplemented with ampicillin and kanamycin. The expression of S. marcescens hemolysin genes in wild-type E. coli and in an hns-defective derivative at different pH and during different growth phases indicated that, in the absence of H-NS, the expression of hemolysin did not vary with pH changes or growth phases. Furthermore, the data suggest that H-NS may play an important role in the regulation of hemolysin expression in S. marcescens and its effect may be due to changes in DNA topology influencing transcription and thus the amount of hemolysin expression. Implications for the mechanism by which H-NS influences gene expression are discussed.