DNA Methylation in the Fields of Prenatal Diagnosis and Early Detection of Cancers.

The central objective of the metamorphosis of discovery science into biomedical applications is to serve the purpose of patients and curtail the global disease burden. The journey from the discovery of DNA methylation (DNAm) as a biological process to its emergence as a diagnostic tool is one of the finest examples of such metamorphosis and has taken nearly a century. Particularly in the last decade, the application of DNA methylation studies in the clinic has been standardized more than ever before, with great potential to diagnose a multitude of diseases that are associated with a burgeoning number of genes with this epigenetic alteration. Fetal DNAm detection is becoming useful for noninvasive prenatal testing, whereas, in very preterm infants, DNAm is also shown to be a potential biological indicator of prenatal risk factors. In the context of cancer, liquid biopsy-based DNA-methylation profiling is offering valuable epigenetic biomarkers for noninvasive early-stage diagnosis. In this review, we focus on the applications of DNA methylation in prenatal diagnosis for delivering timely therapy before or after birth and in detecting early-stage cancers for better clinical outcomes. Furthermore, we also provide an up-to-date commercial landscape of DNAm biomarkers for cancer detection and screening of cancers of unknown origin.

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.

Butyrylation Meets Adipogenesis-Probed by a p300-Catalyzed Acylation-Specific Small Molecule Inhibitor: Implication in Anti-obesity Therapy.

The enzyme p300, besides having acetyltransferase activity, can also catalyze other acylation modifications, whose physiological implications are still being investigated. Here, we report that the level of histone butyrylation increases globally as well as locally in the promoters of pro-adipogenic genes during adipogenesis. To delineate the role of p300-catalyzed butyrylation from acetylation in adipogenesis, we identified a semisynthetic derivative (LTK-14A) of garcinol, which specifically inhibited histone butyrylation without affecting acetylation. Treatment of 3T3L1 cells with LTK-14A abolished adipogenesis with downregulation of pro-adipogenic genes along with inhibition of H4K5 butyrylation. Administering LTK-14A to high-fat diet-fed and genetically obese db/db mice led to attenuation/decrease in their weight gain. The reduced obesity could be partially attributed to the inhibition of H4K5 butyrylation in adipocytes and liver. This report therefore not only, for the first time, causally links histone butyrylation with adipogenesis but also presents a probable candidate for anti-obesity therapeutics.

Regulation of Sox8 through lncRNA Mrhl-Mediated Chromatin Looping in Mouse Spermatogonia.

Sox8 is a developmentally important transcription factor that plays an important role in sex maintenance and fertility of adult mice. In the B-type spermatogonial cells, Sox8 is regulated by the long noncoding RNAs (lncRNA) Mrhl in a p68-dependant manner under the control of the Wnt signaling pathway. The downregulation of Mrhl leads to the meiotic commitment of the spermatogonial cells in a Sox8-dependant manner. While the molecular players involved in the regulation of transcription at the Sox8 promoter have been worked out, our current study points to the involvement of the architectural proteins CTCF and cohesin in mediating a chromatin loop that brings the Sox8 promoter in contact with a silencer element present within the gene body in the presence of lncRNA Mrhl concomitant with transcriptional repression. Further, lncRNA Mrhl interacts with the Sox8 locus through the formation of a DNA:DNA:RNA triplex, which is necessary for the recruitment of PRC2 to the locus. The downregulation of lncRNA Mrhl results in the promoter-silencer loop giving way to a promoter-enhancer loop. This active transcription-associated chromatin loop is mediated by YY1 and brings the promoter in contact with the enhancer present downstream of the gene.

LncRNA Hmrhl regulates expression of cancer related genes in chronic myelogenous leukemia through chromatin association.

Long non-coding RNA has emerged as a key regulator of myriad gene functions. One such lncRNA mrhl, reported by our group, was found to have important role in spermatogenesis and embryonic development in mouse. Recently, its human homolog, Hmrhl was shown to have differential expression in several type of cancers. In the present study, we further characterize molecular features of Hmrhl and gain insight into its functional role in leukemia by gene silencing and transcriptome-based studies. Results indicate its high expression in CML patient samples as well as in K562 cell line. Silencing experiments suggest role of Hmrhl in cell proliferation, migration & invasion. RNA-seq and ChiRP-seq data analysis further revealed its association with important biological processes, including perturbed expression of crucial TFs and cancer-related genes. Among them ZIC1, PDGRFß and TP53 were identified as regulatory targets, with high possibility of triplex formation by Hmrhl at their promoter site. Further, overexpression of PDGRFß in Hmrhl silenced cells resulted in rescue effect of cancer associated cellular phenotypes. In addition, we also found TAL-1 to be a potential regulator of Hmrhl expression in K562 cells. Thus, we hypothesize that Hmrhl lncRNA may play a significant role in the pathobiology of CML.

Ubiquitination, Biotech Startups, and the Future of TRIM Family Proteins: A TRIM-Endous Opportunity.

Ubiquitination is a post-translational modification that has pivotal roles in protein degradation and diversified cellular processes, and for more than two decades it has been a subject of interest in the biotech or biopharmaceutical industry. Tripartite motif (TRIM) family proteins are known to have proven E3 ubiquitin ligase activities and are involved in a multitude of cellular and physiological events and pathophysiological conditions ranging from cancers to rare genetic disorders. Although in recent years many kinds of E3 ubiquitin ligases have emerged as the preferred choices of big pharma and biotech startups in the context of protein degradation and disease biology, from a surface overview it appears that TRIM E3 ubiquitin ligases are not very well recognized yet in the realm of drug discovery. This article will review some of the blockbuster scientific discoveries and technological innovations from the world of ubiquitination and E3 ubiquitin ligases that have impacted the biopharma community, from biotech colossuses to startups, and will attempt to evaluate the future of TRIM family proteins in the province of E3 ubiquitin ligase-based drug discovery.

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.

Genome-wide occupancy reveals the localization of H1T2 (H1fnt) to repeat regions and a subset of transcriptionally active chromatin domains in rat spermatids.

BACKGROUND: H1T2/H1FNT is a germ cell-specific linker histone variant expressed during spermiogenesis specifically in round and elongating spermatids. Infertile phenotype of homozygous H1T2 mutant male mice revealed the essential function of H1T2 for the DNA condensation and histone-to-protamine replacement in spermiogenesis. However, the mechanism by which H1T2 imparts the inherent polarity within spermatid nucleus including the additional protein partners and the genomic domains occupied by this linker histone are unknown. RESULTS: Sequence analysis revealed the presence of Walker motif, SR domains and putative coiled-coil domains in the C-terminal domain of rat H1T2 protein. Genome-wide occupancy analysis using highly specific antibody against the CTD of H1T2 demonstrated the binding of H1T2 to the LINE L1 repeat elements and to a significant percentage of the genic regions (promoter-TSS, exons and introns) of the rat spermatid genome. Immunoprecipitation followed by mass spectrometry analysis revealed the open chromatin architecture of H1T2 occupied chromatin encompassing the H4 acetylation and other histone PTMs characteristic of transcriptionally active chromatin. In addition, the present study has identified the interacting protein partners of H1T2-associated chromatin mainly as nucleo-skeleton components, RNA-binding proteins and chaperones. CONCLUSIONS: Linker histone H1T2 possesses unique domain architecture which can account for the specific functions associated with chromatin remodeling events facilitating the initiation of histone to transition proteins/protamine transition in the polar apical spermatid genome. Our results directly establish the unique function of H1T2 in nuclear shaping associated with spermiogenesis by mediating the interaction between chromatin and nucleo-skeleton, positioning the epigenetically specialized chromatin domains involved in transcription coupled histone replacement initiation towards the apical pole of round/elongating spermatids.

Analysis of cellular models of clonal evolution reveals co-evolution of imatinib and HSP90 inhibitor resistances.

Treatment relapse due to clonal evolution was shown to be an independent factor for poor prognosis in advanced stages of chronic myeloid leukemia. Overcoming secondary resistance arising due to clonal evolution is still an unmet need and lack of adequate pre-clinical models hampers the identification of underlying mechanisms and testing of alternate treatment strategies. The current study thus aimed to create cellular models to study molecular mechanisms underlying clonal evolution and identify strategies to overcome the secondary drug resistance. Analysis of cell lines derived from three independent cell-based screens revealed the co-evolution specifically of imatinib and HSP90 inhibitor (HSP90i) resistances despite their exposure to a single inhibitor alone. Molecular and biochemical characterization of these cell lines revealed additional cytogenetic abnormalities, differential activation of pro-survival signaling molecules and over expression of ABL kinase and HSP90 genes. Importantly, all the imatinib-HSP90i dual resistant cell lines remained sensitive to sorafenib and vorinostat suggesting their utility in treating patients who relapse upon imatinib treatment due to clonal evolution. In addition, we cite similar examples of dual resistance towards various kinase inhibitors and HSP90i in some cell lines that represent solid cancers suggesting co-evolution leading to secondary drug resistance as a pan-cancer phenomenon. Taken together, our results suggest the efficacy of HSP90i in overcoming drug resistance caused by point mutations in the target kinase but not in cases of clonal evolution.

Rise of TRIM8: A Molecule of Duality.

The human tripartite motif containing protein 8 (TRIM8), a member of TRIM family proteins, is known to play a dual role as both tumor suppressor and oncogene, and to function at the crosstalk of cancer and innate immunity. In this review, in addition to accumulating recent corroborations that endorse this dual character of TRIM8, we appraise the game-changing capacity of TRIM8 under stress conditions against the backdrop of cell proliferation, apoptosis, and cancer, and also highlight the duality of TRIM8 in multiple contexts like cellular localization, stress-induced conditions, and E3 ubiquitin ligase activity. Finally, we discuss the emerging role of TRIM8 during bipolar spindle formation and mitotic progression, and its growing sphere of influence across multiple human cancers and pathologies, and suggest TRIM8-linked axes that can be modulated further for anti-cancer therapeutics development.

TH2BS11ph histone mark is enriched in the unsynapsed axes of the XY body and predominantly associates with H3K4me3-containing genomic regions in mammalian spermatocytes.

BACKGROUND: TH2B is a major histone variant that replaces about 80-85% of somatic H2B in mammalian spermatocytes and spermatids. The post-translational modifications (PTMs) on TH2B have been well characterised in spermatocytes and spermatids. However, the biological function(s) of these PTMs on TH2B have not been deciphered in great detail. In our attempt to decipher the unique function(s) of histone variant TH2B, we detected the modification in the N-terminal tail, Serine 11 phosphorylation on TH2B (TH2BS11ph) in spermatocytes. RESULTS: The current study is aimed at understanding the function of the TH2BS11ph modification in the context of processes that occur during meiotic prophase I. Immunofluorescence studies with the highly specific antibodies revealed that TH2BS11ph histone mark is enriched in the unsynapsed axes of the sex body and is associated with XY body-associated proteins like Scp3, γH2AX, pATM, ATR, etc. Genome-wide occupancy studies as determined by ChIP sequencing experiments in P20 C57BL6 mouse testicular cells revealed that TH2BS11ph is enriched in X and Y chromosomes confirming the immunofluorescence staining pattern in the pachytene spermatocytes. Apart from the localisation of this modification in the XY body, TH2BS11ph is majorly associated with H3K4me3-containing genomic regions like gene promoters, etc. These data were also found to corroborate with the ChIP sequencing data of TH2BS11ph histone mark carried out in P12 C57BL6 mouse testicular cells, wherein we found the predominant localisation of this modification at H3K4me3-containing genomic regions. Mass spectrometry analysis of proteins that associate with TH2BS11ph-containing mononucleosomes revealed key proteins linked with the functions of XY body, pericentric heterochromatin and transcription. CONCLUSIONS: TH2BS11ph modification is densely localised in the unsynapsed axes of the XY body of the pachytene spermatocyte. By ChIP sequencing studies in mouse P12 and P20 testicular cells, we demonstrate that TH2BS11ph is predominantly associated with H3K4me3 positive genomic regions like gene promoters, etc. We propose that TH2BS11ph modification could act alone or in concert with other histone modifications to recruit the appropriate transcription or XY body recombination protein machinery at specific genomic loci.

Nonhistone human chromatin protein PC4 is critical for genomic integrity and negatively regulates autophagy.

Multifunctional human transcriptional positive co-activator 4 (PC4) is a bona fide nonhistone component of the chromatin and plays a pivotal role in the process of chromatin compaction and functional genome organization. Knockdown of PC4 expression causes a drastic decompaction which leads to open conformation of the chromatin, and thereby altered nuclear architecture, defects in chromosome segregation and changed epigenetic landscape. Interestingly, these defects do not induce cellular death but result in enhanced cellular proliferation, possibly through enhanced autophagic activity. Moreover, PC4 depletion confers significant resistance to gamma irradiation. Exposure to gamma irradiation further induced autophagy in these cells. Inhibition of autophagy by small molecule inhibitors as well as by silencing of a critical autophagy gene drastically reduces the ability of PC4 knockdown cells to survive. On the contrary, complementation with wild-type PC4 could reverse this phenomenon, confirming the process of autophagy as the key mechanism for radiation resistance in the absence of PC4. These data connect the unexplored role of chromatin architecture in regulating autophagy during stress conditions such as radiation.

A novel enhancer RNA, Hmrhl, positively regulates its host gene, phkb, in chronic myelogenous leukemia.

Noncoding RNAs are increasingly being accredited with key roles in gene regulation during development and disease. Here we report the discovery and characterization of a novel long noncoding RNA, Hmrhl, which shares synteny and partial sequence similarity with the mouse lncRNA, Mrhl. The human homolog, Hmrhl, transcribed from intron 14 of phkb gene, is 5.5 kb in size, expressed in all tissues examined and is associated with chromatin. Analysis of Hmrhl locus using ENCODE database revealed that it exhibits hallmarks of enhancers like the open chromatin configuration, binding of transcription factors, enhancer specific histone signature etc. in the K562 Chronic Myelogenous Leukemia (CML) cells. We compared the expression of Hmrhl in the normal lymphoblast cell line, GM12878, with that of K562 cells and lymphoma samples and show that it is highly upregulated in leukemia as well as several cases of lymphoma. Further, we validated the enhancer properties of Hmrhl locus in K562 cells with the help of ChIP-qPCR and Luciferase assay. Moreover, siRNA mediated down-regulation of Hmrhl in K562 cells leads to a concomitant down regulation of its parent gene, phkb, showing that Hmrhl functions as an enhancer RNA and positively regulates its host gene, phkb, in chronic myelogenous leukemia. This study is significant in view of the fact that a better understanding of mechanism of gene regulation under normal conditions and its perturbation in cancer could in turn help in its therapeutic intervention through molecular medicine/RNA based drug discovery.

Spermatid-specific linker histone HILS1 is a poor condenser of DNA and chromatin and preferentially associates with LINE-1 elements.

BACKGROUND: Linker histones establish and maintain higher-order chromatin structure. Eleven linker histone subtypes have been reported in mammals. HILS1 is a spermatid-specific linker histone, and its expression overlaps with the histone-protamine exchange process during mammalian spermiogenesis. However, the role of HILS1 in spermatid chromatin remodeling is largely unknown. RESULTS: In this study, we demonstrate using circular dichroism spectroscopy that HILS1 is a poor condenser of DNA and chromatin compared to somatic linker histone H1d. Genome-wide occupancy study in elongating/condensing spermatids revealed the preferential binding of HILS1 to the LINE-1 (L1) elements within the intergenic and intronic regions of rat spermatid genome. We observed specific enrichment of the histone PTMs like H3K9me3, H4K20me3 and H4 acetylation marks (H4K5ac and H4K12ac) in the HILS1-bound chromatin complex, whereas H3K4me3 and H3K27me3 marks were absent. CONCLUSIONS: HILS1 possesses significantly lower α-helicity compared to other linker histones such as H1t and H1d. Interestingly, in contrast to the somatic histone variant H1d, HILS1 is a poor condenser of chromatin which demonstrate the idea that this particular linker histone variant may have distinct role in histone to protamine replacement. Based on HILS1 ChIP-seq analysis of elongating/condensing spermatids, we speculate that HILS1 may provide a platform for the structural transitions and forms the higher-order chromatin structures encompassing LINE-1 elements during spermiogenesis.

DDX5/p68 associated lncRNA LOC284454 is differentially expressed in human cancers and modulates gene expression.

Long non-coding RNAs (lncRNAs) are emerging as important players in regulation of gene expression in higher eukaryotes. DDX5/p68 RNA helicase protein which is involved in splicing of precursor mRNAs also interacts with lncRNAs like, SRA and mrhl, to modulate gene expression. We performed RIP-seq analysis in HEK293T cells to identify the complete repertoire of DDX5/p68 interacting transcripts including 73 single exonic (SE) lncRNAs. The LOC284454 lncRNA is the second top hit of the list of SE lncRNAs which we have characterized in detail for its molecular features and cellular functions. The RNA is located in the same primary transcript harboring miR-23a∼27a∼24-2 cluster. LOC284454 is a stable, nuclear restricted and chromatin associated lncRNA. The sequence is conserved only in primates among 26 different species and is expressed in multiple human tissues. Expression of LOC284454 is significantly reduced in breast, prostate, uterus and kidney cancer and also in breast cancer cell lines (MCF7 and T47D). Global gene expression studies upon loss and gain of function of LOC284454 revealed perturbation of genes related to cancer-related pathways. Focal adhesion and cell migration pathway genes are downregulated under overexpression condition, and these genes are significantly upregulated in breast cancer cell lines as well as breast cancer tissue samples suggesting a functional role of LOC284454 lncRNA in breast cancer pathobiology.