H4K16ac activates the transcription of transposable elements and contributes to their cis-regulatory function.

Mammalian genomes harbor abundant transposable elements (TEs) and their remnants, with numerous epigenetic repression mechanisms enacted to silence TE transcription. However, TEs are upregulated during early development, neuronal lineage, and cancers, although the epigenetic factors contributing to the transcription of TEs have yet to be fully elucidated. Here, we demonstrate that the male-specific lethal (MSL)-complex-mediated histone H4 acetylation at lysine 16 (H4K16ac) is enriched at TEs in human embryonic stem cells (hESCs) and cancer cells. This in turn activates transcription of subsets of full-length long interspersed nuclear elements (LINE1s, L1s) and endogenous retrovirus (ERV) long terminal repeats (LTRs). Furthermore, we show that the H4K16ac-marked L1 and LTR subfamilies display enhancer-like functions and are enriched in genomic locations with chromatin features associated with active enhancers. Importantly, such regions often reside at boundaries of topologically associated domains and loop with genes. CRISPR-based epigenetic perturbation and genetic deletion of L1s reveal that H4K16ac-marked L1s and LTRs regulate the expression of genes in cis. Overall, TEs enriched with H4K16ac contribute to the cis-regulatory landscape at specific genomic locations by maintaining an active chromatin landscape at TEs.

Identification of PAX6 and NFAT4 as the Transcriptional Regulators of the Long Noncoding RNA Mrhl in Neuronal Progenitors.

The long noncoding RNA (lncRNA) Mrhl has been shown to be involved in coordinating meiotic commitment of mouse spermatogonial progenitors and differentiation events in mouse embryonic stem cells. Here, we characterized the interplay of Mrhl with lineage-specific transcription factors during mouse neuronal lineage development. Our results demonstrate that Mrhl is expressed in the neuronal progenitor populations in mouse embryonic brains and in retinoic acid-derived radial-glia-like neuronal progenitor cells. Depletion of Mrhl leads to early differentiation of neuronal progenitors to a more committed state. A master transcription factor, PAX6, directly binds to the Mrhl promoter at a major site in the distal promoter, located at 2.9 kb upstream of the transcription start site (TSS) of Mrhl. Furthermore, NFAT4 occupies the Mrhl-proximal promoter at two sites, at 437 base pairs (bp) and 143 bp upstream of the TSS. Independent knockdown studies for PAX6 and NFAT4 confirm that they regulate Mrhl expression in neuronal progenitors. We also show that PAX6 and NFAT4 associate with each other in the same chromatin complex. NFAT4 occupies the Mrhl promoter in PAX6-bound chromatin, implying possible coregulation of Mrhl. Our studies are crucial for understanding how lncRNAs are regulated by major lineage-specific transcription factors, in order to define specific development and differentiation events.

LncRNA Mrhl orchestrates differentiation programs in mouse embryonic stem cells through chromatin mediated regulation.

Long non-coding RNAs (lncRNAs) have been well-established to act as regulators and mediators of development and cell fate specification programs. LncRNA Mrhl (meiotic recombination hotspot locus) has been shown to act in a negative feedback loop with WNT signaling to regulate male germ cell meiotic commitment. In our current study, we have addressed the role of Mrhl in development and differentiation using mouse embryonic stem cells (mESCs) as our model system of study. Mrhl is a nuclear-localized, chromatin-bound lncRNA with moderately stable expression in mESCs. Transcriptome analyses and loss-of-function phenotype studies revealed dysregulation of developmental processes, lineage-specific transcription factors and key networks along with aberrance in specification of early lineages during differentiation of mESCs. Genome-wide chromatin occupancy studies suggest regulation of chromatin architecture at key target loci through triplex formation. Our studies thus reveal a role for lncRNA Mrhl in regulating differentiation programs in mESCs in the context of appropriate cues through chromatin-mediated responses.

Long Noncoding RNA: Genome Organization and Mechanism of Action.

For the last four decades, we have known that noncoding RNAs maintain critical housekeeping functions such as transcription, RNA processing, and translation. However, in the late 1990s and early 2000s, the advent of high-throughput sequencing technologies and computational tools to analyze these large sequencing datasets facilitated the discovery of thousands of small and long noncoding RNAs (lncRNAs) and their functional role in diverse biological functions. For example, lncRNAs have been shown to regulate dosage compensation, genomic imprinting, pluripotency, cell differentiation and development, immune response, etc. Here we review how lncRNAs bring about such copious functions by employing diverse mechanisms such as translational inhibition, mRNA degradation, RNA decoys, facilitating recruitment of chromatin modifiers, regulation of protein activity, regulating the availability of miRNAs by sponging mechanism, etc. In addition, we provide a detailed account of different mechanisms as well as general principles by which lncRNAs organize functionally different nuclear sub-compartments and their impact on nuclear architecture.

Long Noncoding RNAs in Pluripotency of Stem Cells and Cell Fate Specification.

Since the annotation of the mouse genome (FANTOM project) [Kawai J et al (2001) Functional annotation of a full-length mouse cDNA collection. Nature 409(6821):685-690] or the human genome [An integrated encyclopedia of DNA elements in the human genome. (2012) Nature 489(7414):57-74; Harrow J et al (2012) GENCODE: the reference human genome annotation for the ENCODE project. Genome Res 22(9):1760-1774], the roles of long noncoding RNAs in coordinating specific signaling pathways have been established in a wide variety of model systems. They have emerged as crucial and key regulators of stem cell maintenance and/or their differentiation into different lineages. In this chapter we have discussed the recently discovered lncRNAs that have been shown to be necessary for the maintenance of pluripotency of both mouse and human ES cells. We have also highlighted the different lncRNAs which are involved in directed differentiation of stem cells into any of the three germ layers. In recent years stem cell therapies including bone marrow transplantation are becoming an integral part of modern medicinal practices. However, there are still several challenges in making stem cell therapy more reproducible so that the success rate reaches a high percentage in the clinic. It is hoped that understanding the molecular mechanisms pertaining to the role of these newly discovered lncRNAs in the differentiation process of stem cells to specific lineages should pave the way to make stem cell therapy and regenerative medicine as a normal clinical practice in the near future.

Long noncoding RNAs in development and cancer: potential biomarkers and therapeutic targets.

Long noncoding RNAs are emerging as key players in various fundamental biological processes. We highlight the varied molecular mechanisms by which lncRNAs modulate gene expression in diverse cellular contexts and their role in early mammalian development in this review. Furthermore, it is being increasingly recognized that altered expression of lncRNAs is specifically associated with tumorigenesis, tumor progression and metastasis. We discuss various lncRNAs implicated in different cancer types with a focus on their clinical applications as potential biomarkers and therapeutic targets in the pathology of diverse cancers.