Genome-wide screens identify specific drivers of mutant hTERT promoters

Cancer-specific hTERT promoter mutations reported in 19% of cancers result in enhanced telomerase activity. Understanding the distinctions between transcriptional regulation of wild-type (WT) and mutant (Mut) hTERT promoters may open up avenues for development of inhibitors which specially block hTERT expression in cancer cells. To comprehensively identify physiological regulators of WT- or Mut-hTERT promoters, we generated several isogenic reporter cells driven by endogenous hTERT loci. Genome-wide CRISPR-Cas9 and small interfering RNA screens using these isogenic reporter lines identified specific regulators of Mut-hTERT promoters. We validate and characterize one of these hits, namely, MED12, a kinase subunit of mediator complex. We demonstrate that MED12 specifically drives expression of hTERT from the Mut-hTERT promoter by mediating long-range chromatin interaction between the proximal Mut-hTERT promoter and T-INT1 distal regulatory region 260 kb upstream. Several hits identified in our screens could serve as potential therapeutic targets, inhibition of which may specifically block Mut-hTERT promoter driven telomerase reactivation in cancers.

Differential ESR1 Promoter Methylation in the Peripheral Blood—Findings from the Women 40+ Healthy Aging Study

Background Estrogen receptor α (ERα) contributes to maintaining biological processes preserving health during aging. DNA methylation changes of ERα gene (ESR1) were established as playing a direct role in the regulation of ERα levels. In this study, we hypothesized decreased DNA methylation of ESR1 associated with postmenopause, lower estradiol (E2) levels, and increased age among healthy middle-aged and older women. Methods We assessed DNA methylation of ESR1 promoter region from dried blood spots (DBSs) and E2 from saliva samples in 130 healthy women aged 40–73 years. Results We found that postmenopause and lower E2 levels were associated with lower DNA methylation of a distal regulatory region, but not with DNA methylation of proximal promoters. Conclusion Our results indicate that decreased methylation of ESR1 cytosine-phosphate-guanine island (CpGI) shore may be associated with conditions of lower E2 in older healthy women.

MUNC, an Enhancer RNA Upstream from the MYOD Gene, Induces a Subgroup of Myogenic Transcripts in trans Independently of MyoD

ABSTRACT MyoD upstream noncoding RNA (MUNC) initiates in the distal regulatory region (DRR) enhancer of MYOD and is formally classified as an enhancer RNA (DRReRNA). MUNC is required for optimal myogenic differentiation, induces specific myogenic transcripts in trans (MYOD, MYOGENIN, and MYH3), and has a functional human homolog. The vast majority of eRNAs are believed to act in cis primarily on their neighboring genes (1, 2), making it likely that MUNC action is dependent on the induction of MYOD RNA. Surprisingly, MUNC overexpression in MYOD−/− C2C12 cells induces many myogenic transcripts in the complete absence of MyoD protein. Genomewide analysis showed that, while many genes are regulated by MUNC in a MyoD-dependent manner, there is a set of genes that are regulated by MUNC, both upward and downward, independently of MyoD. MUNC and MyoD even appear to act antagonistically on certain transcripts. Deletion mutagenesis showed that there are at least two independent functional sites on the MUNC long noncoding RNA (lncRNA), with exon 1 more active than exon 2 and with very little activity from the intron. Thus, although MUNC is an eRNA of MYOD, it is also a trans-acting lncRNA whose sequence, structure, and cooperating factors, which include but are not limited to MyoD, determine the regulation of many myogenic genes.

MUNC, a Long Noncoding RNA That Facilitates the Function of MyoD in Skeletal Myogenesis

Anin silicoscreen for myogenic long noncoding RNAs (lncRNAs) revealed nine lncRNAs that are upregulated more than 10-fold in myotubes versus levels in myoblasts. One of these lncRNAs, MyoD upstream noncoding (MUNC, also known as DRReRNA), is encoded 5 kb upstream of the transcription start site ofMyoD, a myogenic transcription factor gene. MUNC is specifically expressed in skeletal muscle and exists as in unspliced and spliced isoforms, and its 5′ end overlaps with thecis-acting distal regulatory region (DRR) ofMyoD. Small interfering RNA (siRNA) of MUNC reduced myoblast differentiation and specifically reduced the association of MyoD to the DRR enhancer and myogenin promoter but not to another MyoD-dependent enhancer. Stable overexpression of MUNC from a heterologous promoter increased endogenousMyoD,Myogenin, andMyh3(myosin heavy chain, [MHC] gene) mRNAs but not the cognate proteins, suggesting that MUNC can act intransto promote gene expression but that this activity does not require an induction of MyoD protein. MUNC also stimulates the transcription of other genes that are not recognized as MyoD-inducible genes. Knockdown of MUNCin vivoimpaired murine muscle regeneration, implicating MUNC in primary satellite cell differentiation in the animal. We also discovered a human MUNC that is induced during differentiation of myoblasts and whose knockdown decreases differentiation, suggesting an evolutionarily conserved role of MUNC lncRNA in myogenesis. Although MUNC overlaps with the DRR enhancer, our results suggest that MUNC is not a classiccis-acting enhancer RNA (e-RNA) acting exclusively by stimulating the neighboringMyoDgene but more like a promyogenic lncRNA that acts directly or indirectly on multiple promoters to increase myogenic gene expression.

Mechanism of Action of a Distal NF-κB-Dependent Enhancer

ABSTRACT The monocyte chemoattractant protein 1 gene (MCP-1) is regulated by TNF through an NF-κB-dependent distal enhancer and an Sp1-dependent promoter-proximal regulatory region. In the silent state, only the distal regulatory region is accessible to transcription factors. Upon activation by tumor necrosis factor, NF-κB binds to the distal regulatory region and recruits CBP and p300. CBP and p300 recruitment led to specific histone modifications that ultimately enabled the binding of Sp1 to the proximal regulatory region. During this process, a direct interaction between the distal and proximal regulatory regions occurred. Sp1, NF-κB, CBP, and p300 were required for this interaction. CBP/p300-mediated histone modifications enhanced the binding of the coactivator CARM1 to the distal regulatory region. CARM1, which is necessary for MCP-1 expression, was not required for distal-proximal region interactions, suggesting that it plays a later downstream activation event. The results describe a model in which the separation of the distal enhancer from the promoter-proximal region allows for two independent chromatin states to exist, preventing inappropriate gene activation at the promoter while at the same time allowing rapid induction through the distal regulatory region.

MyoD Distal Regulatory Region Contains an SRF Binding CArG Element Required for MyoD Expression in Skeletal Myoblasts and during Muscle Regeneration

We show here that the distal regulatory region (DRR) of the mouse and human MyoD gene contains a conserved SRF binding CArG-like element. In electrophoretic mobility shift assays with myoblast nuclear extracts, this CArG sequence, although slightly divergent, bound two complexes containing, respectively, the transcription factor YY1 and SRF associated with the acetyltransferase CBP and members of C/EBP family. A single nucleotide mutation in the MyoD-CArG element suppressed binding of both SRF and YY1 complexes and abolished DRR enhancer activity in stably transfected myoblasts. This MyoD-CArG sequence is active in modulating endogeneous MyoD gene expression because microinjection of oligonucleotides corresponding to the MyoD-CArG sequence specifically and rapidly suppressed MyoD expression in myoblasts. In vivo, the expression of a transgenic construct comprising a minimal MyoD promoter fused to the DRR and β-galactosidase was induced with the same kinetics as MyoD during mouse muscle regeneration. In contrast induction of this reporter was no longer seen in regenerating muscle from transgenic mice carrying a mutated DRR-CArG. These results show that an SRF binding CArG element present in MyoD gene DRR is involved in the control of MyoD gene expression in skeletal myoblasts and in mature muscle satellite cell activation during muscle regeneration.