Background Ubiquitously expressed CTCF is involved in numerous cellular functions, such as organizing chromatin into TAD structures. In contrast, its paralog, CTCFL, is normally only present in the testis. However, it is also aberrantly expressed in many cancers. While it is known that shared and unique zinc finger sequences in CTCF and CTCFL enable CTCFL to bind competitively to a subset of CTCF binding sites as well as its own unique locations, the impact of CTCFL on chromosome organization and gene expression has not been comprehensively analyzed in the context of CTCF function. Using an inducible complementation system, we analyze the impact of expressing CTCFL and CTCF-CTCFL chimeric proteins in the presence or absence of endogenous CTCF to clarify the relative and combined contribution of CTCF and CTCFL to chromosome organization and transcription. Results We demonstrate that the N terminus of CTCF interacts with cohesin which explains the requirement for convergent CTCF binding sites in loop formation. By analyzing CTCF and CTCFL binding in tandem, we identify phenotypically distinct sites with respect to motifs, targeting to promoter/intronic intergenic regions and chromatin folding. Finally, we reveal that the N, C, and zinc finger terminal domains play unique roles in targeting each paralog to distinct binding sites to regulate transcription, chromatin looping, and insulation. Conclusion This study clarifies the unique and combined contribution of CTCF and CTCFL to chromosome organization and transcription, with direct implications for understanding how their co-expression deregulates transcription in cancer.

Highlights d HSPCs exhibit post-transcriptional and translational mechanisms of gene regulation d mTOR is targeted for proteasomal degradation in myeloid progenitors by c-Cbl d CDK1 regulates the activation of eIF4E-dependent translation in myeloid progenitors d Aberrant mTOR expression in myeloid progenitors results in increased myeloid cells

BackgroundUbiquitously expressed CTCF is involved in numerous cellular functions, such as organizing chromatin into TAD structures. In contrast, its paralog, CTCFL is normally only present in testis. However, it is also aberrantly expressed in many cancers. While it is known that shared and unique zinc finger sequences in CTCF and CTCFL enable CTCFL to bind competitively to a subset of CTCF binding sites as well as its own unique locations, the impact of CTCFL on chromosome organization and gene expression has not been comprehensively analyzed in the context of CTCF function. Using an inducible complementation system, we analyze the impact of expressing CTCFL and CTCF-CTCFL chimeric proteins in the presence or absence of endogenous CTCF to clarify the relative and combined contribution of CTCF and CTCFL to chromosome organization and transcription.ResultsWe demonstrate that the N terminus of CTCF interacts with cohesin which explains the requirement for convergent CTCF binding sites in loop formation. By analyzing CTCF and CTCFL binding in tandem we identify phenotypically distinct sites with respect to motifs, targeting to promoter/intronic intergenic regions and chromatin folding. Finally, we reveal that the N, C and zinc finger terminal domains play unique roles in targeting each paralog to distinct binding sites, to regulate transcription, chromatin looping and insulation.ConclusionThis study clarifies the unique and combined contribution of CTCF and CTCFL to chromosome organization and transcription, with direct implications for understanding how their co-expression deregulates transcription in cancer.

Epigenetic regulators in normal and malignant hematopoiesis have been shown to be important in normal and malignant stem cell self-renewal function and myeloid leukemogenesis. While epigenetic dysregulation can occur through activating and/or loss-of-function mutations, these regulators can also be modulated by other regulators, such as microRNAs. Specifically, miR-29 has been previously identified as an "epi-mir" for contributing to epigenetic regulation by altering expression of DNMT3a and TET. We and others have previously shown that in the hematopoietic system, miR-29 is a positive regulator of hematopoietic stem cell (HSC) self-renewal, is upregulated in AML blasts, and when over-expressed in transplanted HSCs and immature progenitors, leads to a myeloproliferative-like disorder that progresses to acute myeloid leukemia (AML). To investigate how miR-29 shapes the leukemic stem cell (LSC) epigenome, we transduced the MLL-AF9 fusion oncogene into WT and mir29a/b1 null Lin-ckit+sca1+ (LSK) cells and transplanted them into recipient mice. Transplantation of MLL-AF9+ miR-29 null cells into lethally irradiated recipients resulted in an increased disease latency compared to recipients of WT MLL-AF9+ cells (median: 56 vs 151 days, p<0.001). Characterization of miR-29 null blasts revealed increased expression of myeloid differentiation markers (CD11b, CD14), increased apoptosis, and reduced CFU capacity, consistent with decreased LSC self-renewal. To gain molecular insights into this phenotype, we interrogated RNA-seq data generated from WT and miR-29 null blasts. Compared to WT blasts, miR-29 null blasts showed loss of LSC gene signatures and enrichment for myeloid lineage genes, consistent with increased differentiation (Figure A). In addition, GSEA showed enrichment of H3K27me3, H3K4me2, and gene-specific polycomb group (PcG) associated pathways. On further validation, ChIP-Seq showed overall genome-wide reduction of DNA modification markers such as H3K27me3, H3K9me3, H3K36me3, H3K79me2, and H3K4me3 in miR-29 null blasts compared to WT. Hence, we hypothesized that miR-29 likely targets epigenetic regulators critical for LSC maintenance, and that loss of miR-29 rewires the LSC epigenetic landscape to induce differentiation and abrogate LSC self-renewal. In order to identify downstream mediators of the miR-29 null blast epigenetic phenotype, we used an shRNA library against 500 known epigenetic regulators. The top enriched genes in miR-29 null blasts were members of the PcG family, and as predicted, mRNAs encoding these proteins were upregulated in miR-29 null blasts, including CBX2. These findings were further corroborated by comparing the transcriptomes of AML patient samples to normal hematopoietic stem/progenitor cells (https://gexc.riken.jp), which showed that CBX2 has lower expression in LSCs and blasts compared to normal HSCs and GMPs. When we overexpressed CBX2 in K562 and THP-1 myeloid leukemia cell lines, we observed increased apoptosis and myeloid differentiation as well as impaired clonogenic capacity in vitro, suggesting that downregulation of CBX2 is important in myeloid leukemogenesis. Collectively, our studies indicate that miR-29 promotes LSC function by restricting CBX2 expression to maintain the AML epigenetic landscape. Disclosures No relevant conflicts of interest to declare.

Following publication of the original paper [1], the authors reported an error in the acknowledgements section. The updated acknowledgements section is given below and the changes have been highlighted in bold typeface.

The 2010 Pingry School S.M.A.R.T. Team (Students Modeling A Research Topic) has been working with the Banta laboratory at Columbia University to design and produce accurate, three‐dimensional physical models of alcohol dehydrogenase AdhD and other enzymes with applications for use in a biofuel cell. Features being engineered into these enzymes include (1) self‐assembly into hydrogels, (2) alternate cofactor use, and (3) broader substrate specificity. Discussions with the Banta laboratory allowed the students to use RP‐RasMol to design models of enzymes studied in the lab to highlight their structural and functional characteristics. These designs were used to direct rapid prototyping machines to build physical models of these enzymes. Along with Jmol tutorials created on the Team website (www.pingrysmartteam.com) and in Proteopedia (www.proteopedia.org), these physical models serve as “communication tools” used to enhance the understanding of these enzymes and their applications among the scientific and academic community. By contributing this new tool to the Banta laboratory research team, the students have the unique opportunity to experience and participate in the activities of a research laboratory. This work is supported by a grant awarded to Tim Herman by the NIH NCRR SEPA program and the HHMI Precollege Science Education Program.

Aging hematopoiesis is characterized by increased numbers of immunophenotypic HSCs that exhibit impaired self-renewal and long-term reconstitution potential, both in competitive and noncompetitive settings. We previously demonstrated that normal young mouse HSCs (CD34-CD150+LSK) can be fractionated into subsets based on expression of c-Kit surface expression, with c-Kithi HSCs exhibiting reduced self-renewal and megakaryocytic biased differentiation (Shin et al., 2014). We therefore hypothesized that the expansion of c-Kithi HSCs in old mice could potentially explain the age-related decline in immunophenotypically defined old HSC function. Evaluation of the bone marrow of 24-month-old C57Bl/6 mice revealed that the frequency of c-KithiHSCs (out of total HSCs) is 1.5-fold higher in old mice than in 3-month old mice (P=0.04), while the frequency of c-Kitlo HSCs was 1.5-fold lower in old mice (P=0.007; Fig 1A). This finding is consistent with our previous observation of a megakaryocytic-bias in c-KithiHSCs, since peripheral blood analysis of old mice revealed a 2.1-fold increase in platelets compared to young mice (p<0.01) (Fig 1B). To test the long-term reconstitution potential of aging HSCs, we competitively transplanted 400 c-Kitloor c-Kithi HSCs from 24-month old mice, along with 300,000 competitor bone marrow cells, into lethally irradiated young recipients. Sixteen weeks post-transplantation, mice receiving old c-Kithi HSCs exhibited significantly lower donor peripheral blood chimerism levels compared to old c-Kitlo HSC recipients (9.4% vs 57.1%, P=0.02) (Fig 1C). Both old c-Kithiand old c-Kitlo HSCs exhibited similar myeloid-reconstituting potential (Fig 1D). Furthermore, mice transplanted with old c-Kitlo HSCs exhibited 78% donor HSC chimerism, achieving 6.4-fold higher chimerism levels than mice transplanted with old c-Kithi HSCs, this was comparable to the differences observed with young c-Kitlo and c-Kithi transplanted HSCs (Fig 1E). To quantify the self-renewal capacity of old HSCs, we calculated the "self-renewal quotient" (Challen et al., 2010). This analysis showed that the self-renewal potential in old c-Kithi and c-Kitlo HSCs were 0.8 and 7.8 respectively, indicating higher self-renewal potential in c-Kitlo than c-Kithi HSCs (Fig 1F). Collectively, these data suggest that myeloid-biased differentiation is an age-associated change in hematopoiesis that may not be associated with decreased self-renewal in all HSCs. To gain mechanistic insights underlying these qualitative differences, we interrogated transcriptional profiles of microarray data from c-Kitlo and c-Kithi HSCs, to identify potential pathways critical for HSC maintenance. Gene Ontology and pathway analyses showed several differentially expressed pathways between c-Kithiand c-KitloHSCs, of which genes related to protein translation and mitochondrial activity was significantly enriched in c-Kithi HSCs (Fig 1G). Given the underrepresentation of translation-related genes in c-Kitlo HSCs, we tested whether they exhibit reduced global translation using OP-Puro incorporation assays. These studies confirmed that old c-Kitlo HSCs show lower global translation levels than c-KithiHSCs (Fig 1H). Overall, our studies demonstrate functional heterogeneity among old HSCs and identify a novel strategy to identify old HSCs with preserved self-renewal and long-term reconstitution capacity. The ability to identify and prospectively fractionate old HSCs offers a novel approach to investigate the molecular mechanisms underlying HSC aging. Figure legend. (A) Frequency of c-Kithior c-Kitlo HSCs was assessed by flow cytometry. (B) Circulating platelet numbers were assessed using a Hemavet counter. Competitive transplants of old c-Kitlo and c-Kithi HSCs into lethally irradiated recipients (C-F). Donor chimerism (C) and lineage potential (D) was evaluated in the peripheral blood of primary recipients. Bone marrow was analyzed at 16 weeks, for donor-derived HSC chimerism (E) and self-renewal quotient (F). (G) Enrichment plots comparing microarray data generated from c-Kithiand c-Kitlo HSCs, using pathways translation-related gene sets. (H) OP-Puro incorporation assays in 24-month old mice. Results are representative of three independent experiments, and shown as mean ± SEM. n = 4-5 mice. *, P < 0.05; **, P < 0.01. Figure 1 Disclosures No relevant conflicts of interest to declare.