Angela Garding

Epigenetic Upregulation of lncRNAs at 13q14.3 in Leukemia Is Linked to the In Cis Downregulation of a Gene Cluster That Targets NF-kB

Non-coding RNAs are much more common than previously thought. However, for the vast majority of non-coding RNAs, the cellular function remains enigmatic. The two long non-coding RNA (lncRNA) genes DLEU1 and DLEU2 map to a critical region at chromosomal band 13q14.3 that is recurrently deleted in solid tumors and hematopoietic malignancies like chronic lymphocytic leukemia (CLL). While no point mutations have been found in the protein coding candidate genes at 13q14.3, they are deregulated in malignant cells, suggesting an epigenetic tumor suppressor mechanism. We therefore characterized the epigenetic makeup of 13q14.3 in CLL cells and found histone modifications by chromatin-immunoprecipitation (ChIP) that are associated with activated transcription and significant DNA-demethylation at the transcriptional start sites of DLEU1 and DLEU2 using 5 different semi-quantitative and quantitative methods (aPRIMES, BioCOBRA, MCIp, MassARRAY, and bisulfite sequencing). These epigenetic aberrations were correlated with transcriptional deregulation of the neighboring candidate tumor suppressor genes, suggesting a coregulation in cis of this gene cluster. We found that the 13q14.3 genes in addition to their previously known functions regulate NF-kB activity, which we could show after overexpression, siRNA–mediated knockdown, and dominant-negative mutant genes by using Western blots with previously undescribed antibodies, by a customized ELISA as well as by reporter assays. In addition, we performed an unbiased screen of 810 human miRNAs and identified the miR-15/16 family of genes at 13q14.3 as the strongest inducers of NF-kB activity. In summary, the tumor suppressor mechanism at 13q14.3 is a cluster of genes controlled by two lncRNA genes that are regulated by DNA-methylation and histone modifications and whose members all regulate NF-kB. Therefore, the tumor suppressor mechanism in 13q14.3 underlines the role both of epigenetic aberrations and of lncRNA genes in human tumorigenesis and is an example of colocalization of a functionally related gene cluster.

Gene regulation and priming by topoisomerase IIα in embryonic stem cells

Topoisomerases resolve torsional stress, while their function in gene regulation, especially during cellular differentiation, remains unknown. Here we find that the expression of topo II isoforms, topoisomerase IIα and topoisomerase IIβ, is the characteristic of dividing and postmitotic tissues, respectively. In embryonic stem cells, topoisomerase IIα preferentially occupies active gene promoters. Topoisomerase IIα inhibition compromises genomic integrity, which results in epigenetic changes, altered kinetics of RNA Pol II at target promoters and misregulated gene expression. Common targets of topoisomerase IIα and topoisomerase IIβ are housekeeping genes, while unique targets are involved in proliferation/pluripotency and neurogenesis, respectively. Topoisomerase IIα targets exhibiting bivalent chromatin resolve upon differentiation, concomitant with their activation and occupancy by topoisomerase IIβ, features further observed for long genes. These long silent genes display accessible chromatin in embryonic stem cells that relies on topoisomerase IIα activity. These findings suggest that topoisomerase IIα not only contributes to stem-cell transcriptome regulation but also primes developmental genes for subsequent activation upon differentiation.

CDX2-driven leukemogenesis involves KLF4 repression and deregulated PPARγ signaling

Aberrant expression of the homeodomain transcription factor CDX2 occurs in most cases of acute myeloid leukemia (AML) and promotes leukemogenesis, making CDX2, in principle, an attractive therapeutic target. Conversely, CDX2 acts as a tumor suppressor in colonic epithelium. The effectors mediating the leukemogenic activity of CDX2 and the mechanism underlying its context-dependent properties are poorly characterized, and strategies for interfering with CDX2 function in AML remain elusive. We report data implicating repression of the transcription factor KLF4 as important for the oncogenic activity of CDX2, and demonstrate that CDX2 differentially regulates KLF4 in AML versus colon cancer cells through a mechanism that involves tissue-specific patterns of promoter binding and epigenetic modifications. Furthermore, we identified deregulation of the PPARγ signaling pathway as a feature of CDX2-associated AML and observed that PPARγ agonists derepressed KLF4 and were preferentially toxic to CDX2+ leukemic cells. These data delineate transcriptional programs associated with CDX2 expression in hematopoietic cells, provide insight into the antagonistic duality of CDX2 function in AML versus colon cancer, and suggest reactivation of KLF4 expression, through modulation of PPARγ signaling, as a therapeutic modality in a large proportion of AML patients.

Defective DROSHA processing contributes to downregulation of MiR-15/-16 in chronic lymphocytic leukemia

The MIR-15A/-16-1 tumor suppressor microRNAs (miRNAs) are deleted in leukemic cells from more than 50% of patients with chronic lymphocytic leukemia (CLL). As these miRNAs are also less abundant in patients without genomic deletion, their downregulation in CLL is likely to be caused by additional mechanisms. We found the primary transcripts (pri-miRNAs) of MIR-15a/-16/-15b to be elevated and processing intermediates (precursor miRNAs) to be reduced in cells from CLL patients (22/38) compared with non-malignant B-cells (n=14), indicating a block of miRNA maturation at the DROSHA processing step. Using a luciferase reporter assay for pri-miR processing we validated the defect in primary CLL cells. The block of miRNA maturation is restricted to specific miRNAs and can be found in the cell line MEC-2, but not in MEC-1, even though both are derived from the same CLL patient. In these cells, the RNA-specific deaminase ADARB1 leads to reduced pri-miRNA processing, but full processing efficiency is recovered upon deletion of the RNA-binding domains or nuclear localization of ADARB1. Thus, we show that, apart from genomic deletion or transcriptional downregulation, aberrant processing of miRNA leads to specific reduction of miRNAs in leukemic cells. This represents a novel oncogenic mechanism in the pathogenesis of CLL.

Dynamics and function of distal regulatory elements during neurogenesis and neuroplasticity

Gene regulation in mammals involves a complex interplay between promoters and distal regulatory elements that function in concert to drive precise spatiotemporal gene expression programs. However, the dynamics of the distal gene regulatory landscape and its function in the transcriptional reprogramming that underlies neurogenesis and neuronal activity remain largely unknown. Here, we performed a combinatorial analysis of genome-wide data sets for chromatin accessibility (FAIRE-seq) and the enhancer mark H3K27ac, revealing the highly dynamic nature of distal gene regulation during neurogenesis, which gets progressively restricted to distinct genomic regions as neurons acquire a post-mitotic, terminally differentiated state. We further find that the distal accessible and active regions serve as target sites for distinct transcription factors that function in a stage-specific manner to contribute to the transcriptional program underlying neuronal commitment and maturation. Mature neurons respond to a sustained activity of NMDA receptors by epigenetic reprogramming at a large number of distal regulatory regions as well as dramatic reorganization of super-enhancers. Such massive remodeling of the distal regulatory landscape in turn results in a transcriptome that confers a transient loss of neuronal identity and gain of cellular plasticity. Furthermore, NMDA receptor activity also induces many novel prosurvival genes that function in neuroprotective pathways. Taken together, these findings reveal the dynamics of the distal regulatory landscape during neurogenesis and uncover novel regulatory elements that function in concert with epigenetic mechanisms and transcription factors to generate the transcriptome underlying neuronal development and activity.

The transcriptome of mouse central nervous system myelin

Rapid nerve conduction in the CNS is facilitated by insulation of axons with myelin, a specialized oligodendroglial compartment distant from the cell body. Myelin is turned over and adapted throughout life; however, the molecular and cellular basis of myelin dynamics remains elusive. Here we performed a comprehensive transcriptome analysis (RNA-seq) of myelin biochemically purified from mouse brains at various ages and find a surprisingly large pool of transcripts enriched in myelin. Further computational analysis showed that the myelin transcriptome is closely related to the myelin proteome but clearly distinct from the transcriptomes of oligodendrocytes and brain tissues, suggesting a highly selective incorporation of mRNAs into the myelin compartment. The mRNA-pool in myelin displays maturation-dependent dynamic changes of composition, abundance, and functional associations; however ageing-dependent changes after 6 months were minor. We suggest that this transcript pool enables myelin turnover and the local adaptation of individual pre-existing myelin sheaths.

Mapping gene regulatory circuitry of Pax6 during neurogenesis

Pax6 is a highly conserved transcription factor among vertebrates and is important in various aspects of the central nervous system development. However, the gene regulatory circuitry of Pax6 underlying these functions remains elusive. We find that Pax6 targets a large number of promoters in neural progenitors cells. Intriguingly, many of these sites are also bound by another progenitor factor, Sox2, which cooperates with Pax6 in gene regulation. A combinatorial analysis of Pax6-binding data set with transcriptome changes in Pax6-deficient neural progenitors reveals a dual role for Pax6, in which it activates the neuronal (ectodermal) genes while concurrently represses the mesodermal and endodermal genes, thereby ensuring the unidirectionality of lineage commitment towards neuronal differentiation. Furthermore, Pax6 is critical for inducing activity of transcription factors that elicit neurogenesis and repress others that promote non-neuronal lineages. In addition to many established downstream effectors, Pax6 directly binds and activates a number of genes that are specifically expressed in neural progenitors but have not been previously implicated in neurogenesis. The in utero knockdown of one such gene, Ift74, during brain development impairs polarity and migration of newborn neurons. These findings demonstrate new aspects of the gene regulatory circuitry of Pax6, revealing how it functions to control neuronal development at multiple levels to ensure unidirectionality and proper execution of the neurogenic program.

Dynamics of chromatin accessibility and epigenetic state in response to UV damage.

ABSTRACT Epigenetic mechanisms determine the access of regulatory factors to DNA during events such as transcription and the DNA damage response. However, the global response of histone modifications and chromatin accessibility to UV exposure remains poorly understood. Here, we report that UV exposure results in a genome-wide reduction in chromatin accessibility, while the distribution of the active regulatory mark H3K27ac undergoes massive reorganization. Genomic loci subjected to epigenetic reprogramming upon UV exposure represent target sites for sequence-specific transcription factors. Most of these are distal regulatory regions, highlighting their importance in the cellular response to UV exposure. Furthermore, UV exposure results in an extensive reorganization of super-enhancers, accompanied by expression changes of associated genes, which may in part contribute to the stress response. Taken together, our study provides the first comprehensive resource for genome-wide chromatin changes upon UV irradiation in relation to gene expression and elucidates new aspects of this relationship. Summary: Exposure to UV irradiation causes genome-wide changes in chromatin accessibility and epigenetic state.

JNK‐dependent gene regulatory circuitry governs mesenchymal fate

The epithelial to mesenchymal transition (EMT) is a biological process in which cells lose cell–cell contacts and become motile. EMT is used during development, for example, in triggering neural crest migration, and in cancer metastasis. Despite progress, the dynamics of JNK signaling, its role in genomewide transcriptional reprogramming, and involved downstream effectors during EMT remain largely unknown. Here, we show that JNK is not required for initiation, but progression of phenotypic changes associated with EMT. Such dependency resulted from JNK‐driven transcriptional reprogramming of critical EMT genes and involved changes in their chromatin state. Furthermore, we identified eight novel JNK‐induced transcription factors that were required for proper EMT. Three of these factors were also highly expressed in invasive cancer cells where they function in gene regulation to maintain mesenchymal identity. These factors were also induced during neuronal development and function in neuronal migration in vivo. These comprehensive findings uncovered a kinetically distinct role for the JNK pathway in defining the transcriptome that underlies mesenchymal identity and revealed novel transcription factors that mediate these responses during development and disease.

Krüppel-like factor 4 (KLF4) inactivation in chronic lymphocytic leukemia correlates with promoter DNA-methylation and can be reversed by inhibition of NOTCH signaling

The pathomechanism of chronic lymphocytic leukemia (CLL) is an interplay of genomic and epigenomic aberrations. Next generation sequencing (NGS) identified mutations in CLL that impact on tumor suppressor and oncogenes like NOTCH1.[1][1] In addition, CLL cells harbor genome-wide aberrant DNA-