Chromatin structure and 3D architecture define differential functions of PU.1 cis regulatory elements in human blood cell lineages.

The precise spatio-temporal expression of the hematopoietic ETS transcription factor PU.1 that determines the hematopoietic cell fates is tightly regulated at the chromatin level. However, it remains elusive as to how chromatin signatures are linked to this dynamic expression pattern of PU.1 across blood cell lineages. Here we performed an unbiased and in-depth analysis of the relationship between human PU.1 expression, the presence of trans-acting factors, and 3D architecture at various cis-regulatory elements (CRE) proximal to the PU.1 locus. We identified multiple novel CREs at the upstream region of the gene following an integrative inspection for conserved DNA elements at the chromatin-accessible regions in primary human blood lineages. We showed that a subset of CREs localize within a 10 kb-wide cluster that exhibits that exhibit molecular features of a myeloid-specific super-enhancer involved in mediating PU.1 autoregulation, including open chromatin, unmethylated DNA, histone enhancer marks, transcription of enhancer RNAs, and occupancy of the PU.1 protein itself. Importantly, we revealed the presence of common 35-kb-wide CTCF-bound insulated neighborhood that contains the CRE cluster, forming the chromatin territory for lineage-specific and CRE-mediated chromatin interactions. These include functional CRE-promoter interactions in myeloid and B cells but not in erythroid and T cells. Our findings also provide mechanistic insights into the interplay between dynamic chromatin structure and 3D architecture in defining certain CREs as enhancers or silencers in chromatin regulation of PU.1 expression. The study lays the groundwork for further examination of PU.1 CREs as well as epigenetic regulation in malignant hematopoiesis.

PU.1-c-Jun interaction is crucial for PU.1 function in myeloid development.

The Ets transcription factor PU.1 is essential for inducing the differentiation of monocytes, macrophages, and B cells in fetal liver and adult bone marrow. PU.1 controls hematopoietic differentiation through physical interactions with other transcription factors, such as C/EBPα and the AP-1 family member c-Jun. We found that PU.1 recruits c-Jun to promoters without the AP-1 binding sites. To address the functional importance of this interaction, we generated PU.1 point mutants that do not bind c-Jun while maintaining normal DNA binding affinity. These mutants lost the ability to transactivate a target reporter that requires a physical PU.1-c-Jun interaction, and did not induce monocyte/macrophage differentiation of PU.1-deficient cells. Knock-in mice carrying these point mutations displayed an almost complete block in hematopoiesis and perinatal lethality. While the PU.1 mutants were expressed in hematopoietic stem and early progenitor cells, myeloid differentiation was severely blocked, leading to an almost complete loss of mature hematopoietic cells. Differentiation into mature macrophages could be restored by expressing PU.1 mutant fused to c-Jun, demonstrating that a physical PU.1-c-Jun interaction is crucial for the transactivation of PU.1 target genes required for myeloid commitment and normal PU.1 function in vivo during macrophage differentiation.

NAD Modulates DNA Methylation and Cell Differentiation.

Nutritional intake impacts the human epigenome by directing epigenetic pathways in normal cell development via as yet unknown molecular mechanisms. Consequently, imbalance in the nutritional intake is able to dysregulate the epigenetic profile and drive cells towards malignant transformation. Here we present a novel epigenetic effect of the essential nutrient, NAD. We demonstrate that impairment of DNMT1 enzymatic activity by NAD-promoted ADP-ribosylation leads to demethylation and transcriptional activation of the CEBPA gene, suggesting the existence of an unknown NAD-controlled region within the locus. In addition to the molecular events, NAD- treated cells exhibit significant morphological and phenotypical changes that correspond to myeloid differentiation. Collectively, these results delineate a novel role for NAD in cell differentiation, and indicate novel nutri-epigenetic strategies to regulate and control gene expression in human cells.

Myeloid lncRNA LOUP mediates opposing regulatory effects of RUNX1 and RUNX1-ETO in t(8;21) AML.

The mechanism underlying cell type-specific gene induction conferred by ubiquitous transcription factors as well as disruptions caused by their chimeric derivatives in leukemia is not well understood. Here, we investigate whether RNAs coordinate with transcription factors to drive myeloid gene transcription. In an integrated genome-wide approach surveying for gene loci exhibiting concurrent RNA and DNA interactions with the broadly expressed Runt-related transcription factor 1 (RUNX1), we identified the long noncoding RNA (lncRNA) originating from the upstream regulatory element of PU.1 (LOUP). This myeloid-specific and polyadenylated lncRNA induces myeloid differentiation and inhibits cell growth, acting as a transcriptional inducer of the myeloid master regulator PU.1. Mechanistically, LOUP recruits RUNX1 to both the PU.1 enhancer and the promoter, leading to the formation of an active chromatin loop. In t(8;21) acute myeloid leukemia (AML), wherein RUNX1 is fused to ETO, the resulting oncogenic fusion protein, RUNX1-ETO, limits chromatin accessibility at the LOUP locus, causing inhibition of LOUP and PU.1 expression. These findings highlight the important role of the interplay between cell-type-specific RNAs and transcription factors, as well as their oncogenic derivatives in modulating lineage-gene activation and raise the possibility that RNA regulators of transcription factors represent alternative targets for therapeutic development.

Dual functions of the homeoprotein DLX4 in modulating responsiveness of tumor cells to topoisomerase II-targeting drugs.

Topoisomerase II (TOP2)-targeting poisons such as anthracyclines and etoposide are commonly used for cancer chemotherapy and kill tumor cells by causing accumulation of DNA double-strand breaks (DSB). Several lines of evidence indicate that overexpression of TOP2A, the gene encoding topoisomerase IIα, increases sensitivity of tumor cells to TOP2 poisons, but it is not clear why some TOP2A-overexpressing (TOP2A-High) tumors respond poorly to these drugs. In this study, we identified that TOP2A expression is induced by DLX4, a homeoprotein that is overexpressed in breast and ovarian cancers. Analysis of breast cancer datasets revealed that TOP2A-high cases that also highly expressed DLX4 responded more poorly to anthracycline-based chemotherapy than TOP2A-high cases that expressed DLX4 at low levels. Overexpression of TOP2A alone in tumor cells increased the level of DSBs induced by TOP2 poisons. In contrast, DLX4 reduced the level of TOP2 poison-induced DSBs irrespective of its induction of TOP2A. DLX4 did not stimulate homologous recombination-mediated repair of DSBs. However, DLX4 interacted with Ku proteins, stimulated DNA-dependent protein kinase activity, and increased erroneous end-joining repair of DSBs. Whereas DLX4 did not reduce levels of TOP2 poison-induced DSBs in Ku-deficient cells, DLX4 stimulated DSB repair and reduced the level of TOP2 poison-induced DSBs when Ku was reconstituted in these cells. Our findings indicate that DLX4 induces TOP2A expression but reduces sensitivity of tumor cells to TOP2 poisons by stimulating Ku-dependent repair of DSBs. These opposing activities of DLX4 could explain why some TOP2A-overexpressing tumors are not highly sensitive to TOP2 poisons.

A novel hTERT promoter-driven E1A therapeutic for ovarian cancer.

Currently, an effective gene therapy strategy, which not only retains cancer-specific expression but also limits toxicity, has yet to be developed for ovarian cancer. Mounting reports over the years have shown that human telomerase activity is significantly elevated in cancer cells compared with normal cells. In this study, we evaluated the human telomerase reverse transcriptase (hTERT; T) promoter and showed that it can direct target gene expression preferentially in ovarian cancer cells. However, its promoter (T) activity is much lower than that of cytomegalovirus (CMV), a commonly used nonspecific promoter. To overcome this problem, we have integrated the T promoter into our recently developed VP16-Gal4-WPRE integrated systemic amplifier (VISA) system and dramatically enhanced transgene expression. In addition, to further develop this cancer-specific promoter gene expression system into an applicable therapeutic vector, we expressed E1A (an adenoviral type 5 transcription factor that possesses anticancer properties) through this novel VISA platform. We showed that the T-VISA system specifically targeted the expression of E1A to ovarian cancer cells at a level greater than or comparable with the commonly used CMV promoter, yet remained nearly silent in normal cells, thus making this a suitable gene therapy construct. By using this cancer-specific promoter that limits target gene expression in normal cells/tissues, potential toxicity induced by the CMV promoter would be prevented. More importantly, we showed significant antitumor activity with much less toxicity in animal models through i.v. delivery of T-VISA-E1A:liposomal nanoparticles, suggesting a promising role of T-VISA-E1A for ovarian cancer treatment under a gene therapy setting.