Iannis Talianidis

Five-Vertebrate ChIP-seq Reveals the Evolutionary Dynamics of Transcription Factor Binding

Subtle Variation Despite vast phenotypic differences, vertebrates have many readily recognizable specific cell types, like liver hepatocytes. The gene expression that defines specific cells depends on evolutionarily conserved orthologous transcription factors. Schmidt et al. (p. 1036, published online 8 April) studied the conservation and divergence in the genome-wide binding of two such transcription factors, CEBPA and HNF4A, in livers from human, dog, mouse, short-tailed opossum, and chicken. Although the sequence bound by orthologous transcription factors was similar, the vast majority of binding events were unique to each species. Binding of two liver-specific transcription factors in several vertebrate species reveals complex regulatory evolution. Transcription factors (TFs) direct gene expression by binding to DNA regulatory regions. To explore the evolution of gene regulation, we used chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) to determine experimentally the genome-wide occupancy of two TFs, CCAAT/enhancer-binding protein alpha and hepatocyte nuclear factor 4 alpha, in the livers of five vertebrates. Although each TF displays highly conserved DNA binding preferences, most binding is species-specific, and aligned binding events present in all five species are rare. Regions near genes with expression levels that are dependent on a TF are often bound by the TF in multiple species yet show no enhanced DNA sequence constraint. Binding divergence between species can be largely explained by sequence changes to the bound motifs. Among the binding events lost in one lineage, only half are recovered by another binding event within 10 kilobases. Our results reveal large interspecies differences in transcriptional regulation and provide insight into regulatory evolution.

Acetylation Regulates Transcription Factor Activity at Multiple Levels

CREB-binding protein (CBP) possesses an intrinsic acetyltransferase activity capable of acetylating nucleosomal histones as well as several nonhistone proteins. Here, it is shown that CBP can acetylate hepatocyte nuclear factor-4 (HNF-4), a member of the nuclear hormone receptor family, at lysine residues within the nuclear localization sequence. CBP-mediated acetylation is crucial for the proper nuclear retention of HNF-4, which is otherwise transported out to the cytoplasm via the CRM1 pathway. Acetylation also increases HNF-4 DNA binding activity and its affinity of interaction with CBP itself and is required for target gene activation. The results show that acetylation is a key posttranslational modification that may affect several properties of a transcription factor critical for the execution of its biological functions.

Histone modifications defining active genes persist after transcriptional and mitotic inactivation

We examined various histone modifications across the promoter and the coding regions of constitutively active hepatic genes in G0/G1‐enriched, mitotically arrested and α‐amanitin‐blocked cells. Gene activation correlated with localized histone hyperacetylation, H3‐K4 tri‐ or dimethylation and H3‐K79 dimethylation and localized nucleosome remodeling at the promoter and the 5′ portion of the coding regions. Nucleosomes at more downstream locations were monomethylated at H3‐K4. CBP, PCAF, Brg‐1, SNF2H and FACT were recruited to the coding regions in a gene‐specific manner, in a similarly restricted promoter‐proximal pattern. Elongator, however, associated with the more downstream regions. While all factors were dissociated from the chromatin after transcriptional inactivation by α‐amanitin, the histone modifications remained stable. In mitotic cells, histone modifications on parental nucleosomes were preserved and were regenerated in a transcription‐dependent manner at the newly deposited nucleosomes, as the cells entered the next G1 phase. The findings suggest that histone modifications may function as molecular memory bookmarks for previously active locations of the genome, thus contributing to the maintenance of active chromatin states through cell division.

Dynamics of enhancer-promoter communication during differentiation-induced gene activation.

We analyzed the order of recruitment of factors to the HNF-4alpha regulatory regions upon the initial activation of the gene during enterocyte differentiation. An initially independent assembly of regulatory complexes at the proximal promoter and the upstream enhancer regions was followed by the tracking of the entire DNA-protein complex formed on the enhancer along the intervening DNA until it encountered the proximal promoter. This movement correlated with a unidirectional spreading of histone hyperacetylation. Transcription initiation coincided with the formation of a stable enhancer-promoter complex and remodeling of the nucleosome situated at the transcription start site. The results provide experimental evidence for the involvement of a dynamic process culminating in enhancer-promoter communication during long-distance gene activation.

Gene-Specific Modulation of TAF10 Function by SET9-Mediated Methylation

SET9 is a member of the SET domain-containing histone methyltransferase family that can specifically methylate histone 3 at lysine 4 position. Although nucleosomal histones are poor substrates for SET9, the active enzyme can stimulate activator-induced transcription. Here, we show that SET9 can monomethylate the TBP-associated factor TAF10 at a single lysine residue located at the loop 2 region within the putative histone-fold domain of the protein. Methylated TAF10 has an increased affinity for RNA polymerase II, pointing to a direct role of this modification in preinitiation complex formation. Reporter assays and studies on TAF10 null F9 cells expressing a methylation-deficient TAF10 mutant revealed that SET9-mediated methylation of TAF10 potentiates transcription of some but not all TAF10-dependent genes. This gene specificity correlated with SET9 recruitment. The promoter-specific effects of SET9-methylated TAF10 may have important implications regarding the biological function of SET domain-containing lysine methylases, whose primary targets have been presumed to be histones.

Lysine Methylation Regulates E2F1-Induced Cell Death

Histone-modifying enzymes can regulate DNA damage-induced apoptosis through modulation of p53 function. Here, we show that, in p53-deficient tumor cells, Set9 and LSD1 regulate DNA damage-induced cell death in a manner opposite to that observed in p53(+/+) cells, via modulation of E2F1 stabilization. Set9 methylates E2F1 at lysine-185, which prevents E2F1 accumulation during DNA damage and activation of its proapoptotic target gene p73. This methyl mark is removed by LSD1, which is required for E2F1 stabilization and apoptotic function. The molecular mechanism involves crosstalks between lysine methylation and other covalent modifications that affect E2F1 stability. Methylation at lysine-185 inhibits acetylation and phosphorylation at distant positions and, in parallel, stimulates ubiquitination and degradation of the protein. The findings illustrate that the function of methyltransferases can have opposing biological outcomes depending on the specificity of transcription factor targets.

Regulatory Mechanisms Controlling Human Hepatocyte Nuclear Factor 4α Gene Expression

ABSTRACT Hepatocyte nuclear factor 4α (HNF-4α) (nuclear receptor 2A1) is an essential regulator of hepatocyte differentiation and function. Genetic and molecular evidence suggests that the tissue-restricted expression of HNF-4α is regulated mainly at the transcriptional level. As a step toward understanding the molecular mechanism involved in the transcriptional regulation of the human HNF-4α gene, we cloned and analyzed a 12.1-kb fragment of its upstream region. Major DNase I-hypersensitive sites were found at the proximal promoter, the first intron, and the more-upstream region comprising kb −6.5, −8.0, and −8.8. By the use of reporter constructs, we found that the proximal-promoter region was sufficient to drive high levels of hepatocyte-specific transcription in transient-transfection assays. DNase I footprint analysis and electrophoretic mobility shift experiments revealed binding sites for HNF-1α and -β, Sp-1, GATA-6, and HNF-6. High levels of HNF-4α promoter activity were dependent on the synergism between either HNF-1α and HNF-6 or HNF-1β and GATA-6, which implies that at least two alternative mechanisms may activate HNF-4α gene transcription. Chromatin immunoprecipitation experiments with human hepatoma cells showed stable association of HNF-1α, HNF-6, Sp-1, and COUP-TFII with the promoter. The last factor acts as a repressor via binding to a newly identified direct repeat 1 (DR-1) sequence of the human promoter, which is absent in the mouse homologue. We present evidence that this sequence is a bona fide retinoic acid response element and that HNF-4α expression is upregulated in vivo upon retinoic acid signaling.

Regulation of hepatic metabolic pathways by the orphan nuclear receptor SHP

SHP (small heterodimer partner) is an important component of the feedback regulatory cascade, which controls the conversion of cholesterol to bile acids. In order to identify the bona fide molecular targets of SHP, we performed global gene expression profiling combined with chromatin immunoprecipitation assays in transgenic mice constitutively expressing SHP in the liver. We demonstrate that SHP affects genes involved in diverse biological pathways, and in particular, several key genes involved in consecutive steps of cholesterol degradation, bile acid conjugation, transport and lipogenic pathways. Sustained expression of SHP leads to the depletion of hepatic bile acid pool and a concomitant accumulation of triglycerides in the liver. The mechanism responsible for this phenotype includes SHP‐mediated direct repression of downstream target genes and the bile acid sensor FXRα, and an indirect activation of PPARγ and SREBP‐1c genes. We present evidence for the role of altered chromatin configurations in defining distinct gene‐specific mechanisms by which SHP mediates differential transcriptional repression. The multiplicity of genes under its control suggests that SHP is a pleiotropic regulator of diverse metabolic pathways.

Transcription factor-dependent regulation of CBP and P/CAF histone acetyltransferase activity.

CREB‐binding protein (CBP) and CBP‐associated factor (P/CAF) are coactivators possessing an intrinsic histone acetyltransferase (HAT) activity. They are positioned at promoter regions via association with sequence‐specific DNA‐binding factors and stimulate transcription in a gene‐specific manner. The current view suggests that coactivator function depends mainly on the strength and specificity of transcription factor–coactivator interactions. Here we show that two dominant‐negative mutants of hepatocyte nuclear factor‐1α (HNF‐1α), P447L and P519L, occurring in maturity onset diabetes of the young (MODY3) patients, exhibit paradoxically stronger interactions than the wild‐type protein with either CBP or P/CAF. However, CBP and P/CAF recruited by these mutants lack HAT activity. In contrast, wild‐type HNF‐1α and other transcription factors, such as Sp1 or HNF‐4, stimulated the HAT activity of CBP. The results suggest a more dynamic role for DNA‐binding proteins in the transcription process than was considered previously. They are not only required for the recruitment of coactivators to the promoter but they may also modulate their enzymatic activity.

The Intracellular Localization of Deoxycytidine Kinase

Deoxycytidine kinase (dCK) catalyzes the rate-limiting step of the deoxynucleoside salvage pathway in mammalian cells and plays a key role in the activation of several pharmacologically important nucleoside analogs. Using a highly specific polyclonal antibody raised against a C-terminal peptide of the human dCK, we analyzed its subcellular localization by Western blots of biochemically fractionated nuclear and cytoplasmic fractions as well as by in situ immunochemistry. Native dCK was found to be located mainly in the cytoplasm in several cell types, and the enzyme was more concentrated in the perinuclear and cellular membrane area. In contrast, when dCK was overexpressed in the cells, it was mainly located in the nucleus. The results demonstrate that native dCK is a cytoplasmic enzyme. However, it has the ability to enter the nucleus under certain conditions, suggesting the existence of a cytoplasmic retention mechanism that may have an important function in the regulation of the deoxynucleoside salvage pathway.