Identification and Functional Characterization of Two Noncoding RNAs Transcribed from Putative Active Enhancers in Hepatocellular Carcinoma.

Enhancers have been conventionally perceived as cis-acting elements that provide binding sites for trans-acting factors. However, recent studies have shown that enhancers are transcribed and that these transcripts, called enhancer RNAs (eRNAs), have a regulatory function. Here, we identified putative eRNAs by profiling and determining the overlap between noncoding RNA expression loci and eRNA-associated histone marks such as H3K27ac and H3K4me1 in hepatocellular carcinoma (HCC) cell lines. Of the 132 HCC-derived noncoding RNAs, 74 overlapped with the eRNA loci defined by the FANTOM consortium, and 65 were located in the proximal regions of genes differentially expressed between normal and tumor tissues in TCGA dataset. Interestingly, knockdown of two selected putative eRNAs, THUMPD3-AS1 and LINC01572, led to downregulation of their target mRNAs and to a reduction in the proliferation and migration of HCC cells. Additionally, the expression of these two noncoding RNAs and target mRNAs was elevated in tumor samples in the TCGA dataset, and high expression was associated with poor survival of patients. Collectively, our study suggests that noncoding RNAs such as THUMPD3-AS1 and LINC01572 (i.e., putative eRNAs) can promote the transcription of genes involved in cell proliferation and differentiation and that the dysregulation of these noncoding RNAs can cause cancers such as HCC.

Core promoter activity contributes to chromatin-based regulation of internal cryptic promoters.

During RNA polymerase II (RNA Pol II) transcription, the chromatin structure undergoes dynamic changes, including opening and closing of the nucleosome to enhance transcription elongation and fidelity. These changes are mediated by transcription elongation factors, including Spt6, the FACT complex, and the Set2-Rpd3S HDAC pathway. These factors not only contribute to RNA Pol II elongation, reset the repressive chromatin structures after RNA Pol II has passed, thereby inhibiting aberrant transcription initiation from the internal cryptic promoters within gene bodies. Notably, the internal cryptic promoters of infrequently transcribed genes are sensitive to such chromatin-based regulation but those of hyperactive genes are not. To determine why, the weak core promoters of genes that generate cryptic transcripts in cells lacking transcription elongation factors (e.g. STE11) were replaced with those from more active genes. Interestingly, as core promoter activity increased, activation of internal cryptic promoter dropped. This associated with loss of active histone modifications at the internal cryptic promoter. Moreover, environmental changes and transcription elongation factor mutations that downregulated the core promoters of highly active genes concomitantly increased their cryptic transcription. We therefore propose that the chromatin-based regulation of internal cryptic promoters is mediated by core promoter strength as well as transcription elongation factors.

NuA3 HAT antagonizes the Rpd3S and Rpd3L HDACs to optimize mRNA and lncRNA expression dynamics.

In yeast, NuA3 histone acetyltransferase (NuA3 HAT) promotes acetylation of histone H3 lysine 14 (H3K14) and transcription of a subset of genes through interaction between the Yng1 plant homeodomain (PHD) finger and H3K4me3. Although NuA3 HAT has multiple chromatin binding modules with distinct specificities, their interdependence and combinatorial actions in chromatin binding and transcription remain unknown. Modified peptide pulldown assays reveal that the Yng1 N-terminal region is important for the integrity of NuA3 HAT by mediating the interaction between core subunits and two methyl-binding proteins, Yng1 and Pdp3. We further uncover that NuA3 HAT contributes to the regulation of mRNA and lncRNA expression dynamics by antagonizing the histone deacetylases (HDACs) Rpd3S and Rpd3L. The Yng1 N-terminal region, the Nto1 PHD finger and Pdp3 are important for optimal induction of mRNA and lncRNA transcription repressed by the Set2-Rpd3S HDAC pathway, whereas the Yng1 PHD finger-H3K4me3 interaction affects transcriptional repression memory regulated by Rpd3L HDAC. These findings suggest that NuA3 HAT uses distinct chromatin readers to compete with two Rpd3-containing HDACs to optimize mRNA and lncRNA expression dynamics.

Rpd3L HDAC links H3K4me3 to transcriptional repression memory.

Transcriptional memory is critical for the faster reactivation of necessary genes upon environmental changes and requires that the genes were previously in an active state. However, whether transcriptional repression also displays 'memory' of the prior transcriptionally inactive state remains unknown. In this study, we show that transcriptional repression of ∼540 genes in yeast occurs much more rapidly if the genes have been previously repressed during carbon source shifts. This novel transcriptional response has been termed transcriptional repression memory (TREM). Interestingly, Rpd3L histone deacetylase (HDAC), targeted to active promoters induces TREM. Mutants for Rpd3L exhibit increased acetylation at active promoters and delay TREM significantly. Surprisingly, the interaction between H3K4me3 and Rpd3L via the Pho23 PHD finger is critical to promote histone deacetylation and TREM by Rpd3L. Therefore, we propose that an active mark, H3K4me3 enriched at active promoters, instructs Rpd3L HDAC to induce histone deacetylation and TREM.

Modulation of gene expression dynamics by co-transcriptional histone methylations.

Co-transcriptional methylations of histone H3 at lysines 4 and 36, highly conserved methyl marks from yeast to humans, have profound roles in regulation of histone acetylation. These modifications function to recruit and/or activate distinct histone acetyltransferases (HATs) or histone deacetylases (HDACs). Whereas H3K4me3 increases acetylation at promoters via multiple HATs, H3K4me2 targets Set3 HDAC to deacetylate histones in 5' transcribed regions. In 3' regions of genes, H3K36me2/3 facilitates deacetylation by Rpd3S HDAC and slows elongation. Despite their important functions in deacetylation, no strong effects on global gene expression have been seen under optimized or laboratory growth conditions. Instead, H3K4me2-Set3 HDAC and Set2-Rpd3S pathways primarily delay the kinetics of messenger RNA (mRNA) and long noncoding RNA (lncRNA) induction upon environmental changes. A majority of mRNA genes regulated by these pathways have an overlapping lncRNA transcription either from an upstream or an antisense promoter. Surprisingly, the distance between mRNA and lncRNA promoters seems to specify the repressive effects of the two pathways. Given that co-transcriptional methylations and acetylation have been linked to many cancers, studying their functions in a dynamic condition or during cancer progression will be much more important and help identify novel genes associated with cancers.