The transcriptional regulator BCL6 participates in the secondary gene regulatory response to vitamin D.

The vitamin D metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is the high affinity ligand of the transcription factor vitamin D receptor (VDR) and therefore a direct regulator of transcription. Transcriptome-wide analysis of THP-1 human monocytes had indicated more than 600 genes to be significantly (p<0.05) stimulated after 4h incubation with 1,25(OH)2D3, but only 67 of them where more than 1.5-fold up-regulated. These include the genes encoding for the transcription factors BCL6, NFE2, POU4F2 and ELF4, which are controlled by one or two VDR binding sites within their chromosomal domains. The latter are defined via DNA loop formation mediated by the transcription factor CTCF that is highly conserved in its genome-wide loci. We found BCL6 being most responsive to 1,25(OH)2D3 and selected it for further analysis. An incubation of THP-1 cells with 1,25(OH)2D3 for 24 h resulted in a significant (p<0.001) change in the mRNA expression of more than 1600 genes, of which 132 were at least 2-fold up-regulated. About half of the latter genes are secondary 1,25(OH)2D3 targets, since they do not carry any VDR binding site within their chromosomal domain. Chromatin immunoprecipitation sequencing datasets indicated that the majority of these domains contain a BCL6 binding site. We followed the secondary transcriptional response to 1,25(OH)2D3 for eight representative gene examples and confirmed the binding of CTCF and BCL6 to their respective chromosomal domains. In conclusion, our study indicated that in monocytes most of the physiological responses to 1,25(OH)2D3 involve the action of the transcription factor BCL6.

Kaposi's Sarcoma-Associated Herpesvirus Reactivation by Targeting of a dCas9-Based Transcription Activator to the ORF50 Promoter.

CRISPR activation (CRISPRa) has revealed great potential as a tool to modulate the expression of targeted cellular genes. Here, we successfully applied the CRISPRa system to trigger the Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation in latently infected cells by selectively activating ORF50 gene directly from the virus genome. We found that a nuclease-deficient Cas9 (dCas9) fused to a destabilization domain (DD) and 12 copies of the VP16 activation domain (VP192) triggered a more efficient KSHV lytic cycle and virus production when guided to two different sites on the ORF50 promoter, instead of only a single site. To our surprise, the virus reactivation induced by binding of the stable DD-dCas9-VP192 on the ORF50 promoter was even more efficient than reactivation induced by ectopic expression of ORF50. This suggests that recruitment of additional transcriptional activators to the ORF50 promoter, in addition to ORF50 itself, are needed for the efficient virus production. Further, we show that CRISPRa can be applied to selectively express the early lytic gene, ORF57, without disturbing the viral latency. Therefore, CRISPRa-based systems can be utilized to facilitate virus-host interaction studies by controlling the expression of not only cellular but also of specific KSHV genes.

Oncogenic Herpesvirus Engages Endothelial Transcription Factors SOX18 and PROX1 to Increase Viral Genome Copies and Virus Production.

Kaposi sarcoma is a tumor caused by Kaposi sarcoma herpesvirus (KSHV) infection and is thought to originate from lymphatic endothelial cells (LEC). While KSHV establishes latency in virtually all susceptible cell types, LECs support spontaneous expression of oncogenic lytic genes, high viral genome copies, and release of infectious virus. It remains unknown the contribution of spontaneous virus production to the expansion of KSHV-infected tumor cells and the cellular factors that render the lymphatic environment unique to KSHV life cycle. We show here that expansion of the infected cell population, observed in LECs, but not in blood endothelial cells, is dependent on the spontaneous virus production from infected LECs. The drivers of lymphatic endothelium development, SOX18 and PROX1, regulated different steps of the KSHV life cycle. SOX18 enhanced the number of intracellular viral genome copies and bound to the viral origins of replication. Genetic depletion or chemical inhibition of SOX18 caused a decrease of KSHV genome copy numbers. PROX1 interacted with ORF50, the viral initiator of lytic replication, and bound to the KSHV genome in the promoter region of ORF50, increasing its transactivation activity and KSHV spontaneous lytic gene expression and infectious virus release. In Kaposi sarcoma tumors, SOX18 and PROX1 expression correlated with latent and lytic KSHV protein expression. These results demonstrate the importance of two key transcriptional drivers of LEC fate in the regulation of the tumorigenic KSHV life cycle. Moreover, they introduce molecular targeting of SOX18 as a potential novel therapeutic avenue in Kaposi sarcoma. SIGNIFICANCE: SOX18 and PROX1, central regulators of lymphatic development, are key factors for KSHV genome maintenance and lytic cycle in lymphatic endothelial cells, supporting Kaposi sarcoma tumorigenesis and representing attractive therapeutic targets.

Primary Vitamin D Target Genes of Human Monocytes.

The molecular basis of vitamin D signaling implies that the metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) of the secosteroid vitamin D3 activates the transcription factor vitamin D receptor (VDR), which in turn modulates the expression of hundreds of primary vitamin D target genes. Since the evolutionary role of nuclear receptors, such as VDR, was the regulation of cellular metabolism, the control of calcium metabolism became the primary function of vitamin D and its receptor. Moreover, the nearly ubiquitous expression of VDR enabled vitamin D to acquire additional physiological functions, such as the support of the innate immune system in its defense against microbes. Monocytes and their differentiated phenotypes, macrophages and dendritic cells, are key cell types of the innate immune system. Vitamin D signaling was most comprehensively investigated in THP-1 cells, which are an established model of human monocytes. This includes the 1,25(OH)2D3-modulated cistromes of VDR, the pioneer transcription factors PU.1 and CEBPA and the chromatin modifier CTCF as well as of the histone markers of promoter and enhancer regions, H3K4me3 and H3K27ac, respectively. These epigenome-wide datasets led to the development of our chromatin model of vitamin D signaling. This review discusses the mechanistic basis of 189 primary vitamin D target genes identified by transcriptome-wide analysis of 1,25(OH)2D3-stimulated THP-1 cells and relates the epigenomic basis of four different regulatory scenarios to the physiological functions of the respective genes.

Modulation of vitamin D signaling by the pioneer factor CEBPA.

The myeloid master regulator CCAAT enhancer-binding protein alpha (CEBPA) is known as a pioneer factor. In this study, we report the CEBPA cistrome of THP-1 human monocytes after stimulation with the vitamin D receptor (VDR) ligand 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) for 2, 8 and 24 h. About a third of the genomic VDR binding sites co-located with those of CEBPA. In parallel, the binding strength of 5% of the CEBPA cistrome, i.e. some 1500 sites, is significantly (p < 0.001) affected by 1,25(OH)2D3. Transcriptome-wide analysis after CEBPA silencing indicated that the pioneer factor enhances both the basal expression and ligand inducibility of 70 vitamin D target genes largely involved in lipid signaling and metabolism. In contrast, CEBPA suppresses 82 vitamin D target genes many of which are related to the modulation of T cell activity by monocytes. The inducibility of the promoter-specific histone marker H3K4me3 distinguishes the former class of genes from the latter. Moreover, prominent occupancy of the myeloid pioneer factor PU.1 on 1,25(OH)2D3-sensitive CEBPA enhancers mechanistically explains the dichotomy of vitamin D target genes. In conclusion, CEBPA supports vitamin D signaling concerning actions of the innate immune system, but uses the antagonism with PU.1 for suppressing possible overreactions of adaptive immunity.

The impact of the vitamin D-modulated epigenome on VDR target gene regulation.

The micronutrient vitamin D significantly modulates the human epigenome via enhancing genome-wide the rate of accessible chromatin and vitamin D receptor (VDR) binding. This study focuses on histone marks of active chromatin at promoter and enhancer regions and investigates, whether these genomic loci are sensitive to vitamin D. The epigenome of THP-1 human monocytes contains nearly 23,000 sites with H3K4me3 histone modifications, 550 of which sites are significantly (p < 0.05) modulated by stimulation with the VDR ligand 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). H3K27ac histone modifications mark active chromatin and 2473 of 45,500 sites are vitamin D sensitive. The two types of ligand-dependent histone marks allow to distinguish promoter and enhancer regulation by vitamin D, respectively. Transcription start site overlap is the prime attribute of ligand-dependent H3K4me3 marks, while VDR co-location is the top ranking parameter describing 1,25(OH)2D3-sensitive H3K27ac marks at enhancers. A categorization of 1,25(OH)2D3-sensitive histone marks by machine learning algorithms - using the attributes overall peak strength and ligand inducibility - highlights 260 and 287 regions with H3K4me3 and H3K27ac modifications, respectively. These loci are found at the promoter regions of 59 vitamin D target genes and their associated enhancers. In this way, ligand-dependent histone marks provide a link of the effects of 1,25(OH)2D3 on the epigenome with previously reported mRNA expression changes of vitamin D target genes. In conclusion, the human epigenome responds also on the level of histone modifications to 1,25(OH)2D3 stimulation. This allows a more detailed understanding of vitamin D target gene regulation.

The vitamin D-dependent transcriptome of human monocytes.

Monocytes are important cells of the innate immune system that can differentiate into macrophages and dendritic cells. The biologically active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), serves as a ligand of the nuclear receptor vitamin D receptor (VDR). A key physiological function of 1,25(OH)2D3 is the defense against pathogens, such as those causing tuberculosis, that involves the modulation of the monocyte transcriptome. THP-1 cells are an established model of human monocytes, for which the at present largest set of 1,25(OH)2D3-affected genome-wide data are available. Here we summarize the insight obtained from the recent transcriptome of 1,25(OH)2D3-stimulated THP-1 cells, that was determined by triplicate RNA sequencing (RNA-seq). Primary and secondary vitamin D target genes being up- and down-regulated were related to changes in the epigenome of THP-1 cells, such as 1,25(OH)2D3-dependent chromatin opening and modulation of the genome-wide association of the transcription factors VDR and CCCTC-binding factor (CTCF) with their respective genomic binding sites. The anti-microbial response is the top-ranking early physiological function represented by 1,25(OH)2D3-stimulated genomic regions and genes, but also other immunity-related pathways, such as IL10 signaling, are activated. Taken together, the epigenomic and transcriptomic responses of THP-1 cells to 1,25(OH)2D3 represent a master example of the impact of vitamin D on human physiology.