Wen Jin

PML/RARα Targets Promoter Regions Containing PU.1 Consensus and RARE Half Sites in Acute Promyelocytic Leukemia

PML/RARalpha is of crucial importance in acute promyelocytic leukemia (APL) both pathologically and therapeutically. Using a genome-wide approach, we identified in vivo PML/RARalpha binding sites in a PML/RARalpha-inducible cell model. Of the 2979 targeted regions, >62% contained canonical PU.1 motifs and >84% of these PU.1 motifs coexisted with one or more RARE half (RAREh) sites in nearby regions. Promoters with such PU.1-RAREh binding sites were transactivated by PU.1. PU.1-mediated transactivation was repressed by PML/RARalpha and restored by the addition of all-trans retinoic acid (ATRA). Genes containing such promoters were significantly represented by genes transcriptionally suppressed in APL and/or reactivated upon treatment with ATRA. Thus, selective targeting of PU.1-regulated genes by PML/RARalpha is a critical mechanism for the pathogenesis of APL.

An Interferon-Related Signature in the Transcriptional Core Response of Human Macrophages to Mycobacterium tuberculosis Infection

The W-Beijing family of Mycobacterium tuberculosis (Mtb) strains is known for its high-prevalence and -virulence, as well as for its genetic diversity, as recently reported by our laboratories and others. However, little is known about how the immune system responds to these strains. To explore this issue, here we used reverse engineering and genome-wide expression profiling of human macrophage-like THP-1 cells infected by different Mtb strains of the W-Beijing family, as well as by the reference laboratory strain H37Rv. Detailed data mining revealed that host cell transcriptome responses to H37Rv and to different strains of the W-Beijing family are similar and overwhelmingly induced during Mtb infections, collectively typifying a robust gene expression signature (“THP1r2Mtb-induced signature”). Analysis of the putative transcription factor binding sites in promoter regions of genes in this signature identified several key regulators, namely STATs, IRF-1, IRF-7, and Oct-1, commonly involved in interferon-related immune responses. The THP1r2Mtb-induced signature appeared to be highly relevant to the interferon-inducible signature recently reported in active pulmonary tuberculosis patients, as revealed by cross-signature and cross-module comparisons. Further analysis of the publicly available transcriptome data from human patients showed that the signature appears to be relevant to active pulmonary tuberculosis patients and their clinical therapy, and be tuberculosis specific. Thus, our results provide an additional layer of information at the transcriptome level on mechanisms involved in host macrophage response to Mtb, which may also implicate the robustness of the cellular defense system that can effectively fight against genetic heterogeneity in this pathogen.

AML1-ETO targets and suppresses cathepsin G, a serine protease, which is able to degrade AML1-ETO in t(8;21) acute myeloid leukemia.

Although the significance of cathepsin G (CTSG) in host defense has been intensively investigated, little is known about its potential roles in granulopoiesis or leukemogenesis. We report here that CTSG is directly targeted and suppressed by AML1-ETO in t(8;21) acute myeloid leukemia (AML). Luciferase assays demonstrate that the CTSG promoter is strongly transactivated by AML1 and the AML1-dependent transactivation is suppressed by AML1-ETO. We also define a novel regulatory mechanism by which AML1-ETO-mediated transrepression requires both AML1-ETO and AML1 binding at adjacent sites, instead of the replacement of AML1 by AML1-ETO, and wild-type AML1 binding is a prerequisite for the repressive effect caused by AML1-ETO. Further evidence shows that CTSG, as a hematopoietic serine protease, can degrade AML1-ETO both in vitro and in vivo. Restoration of CTSG induces partial differentiation, growth inhibition and apoptosis in AML1-ETO-positive cells. In addition to t(8;21) AML, CTSG downregulation is observed in AML patients with other cytogenetic/genetic abnormalities that potentially interrupt normal AML1 function, that is, inv(16) and EVI1 overexpression. Thus, the targeting and suppression of CTSG by AML1-ETO in t(8;21) AML may provide a mechanism for leukemia cells to escape from the intracellular surveillance system by preventing degradation of foreign proteins.

Structurally differentiated cis-elements that interact with PU.1 are functionally distinguishable in acute promyelocytic leukemia

BackgroundTranscription factor PU.1, a member of the ETS family, is a master regulator of myeloid differentiation whose functional disruption is often associated with acute myeloid leukemia (AML). Although much has been learned about PU.1 over the past decades, relatively little is known about cis-elements that interact with this factor under physiological or pathological conditions, especially in the whole-genome scale. We aimed to define the cistrome of PU.1 in acute promyelocytic leukemia (APL) cells and characterize the cis-elements bound by PU.1.MethodsChromatin immunoprecipitation with specific antibody coupled with deep sequencing (ChIP-seq) was used to investigate the in vivo PU.1 binding sites at the whole-genome scale in APL-derived NB4 cells. The ChIP-quantitative (q)-PCR and luciferase reporter assays were used to validate the binding events and trans-activity, respectively. Various computational analyses, including motif mining, evolutionary conservation analysis and functional enrichment analysis, were performed to characterize the cis-elements that interacted with PU.1.ResultsA total of 26,907 significantly enriched binding regions of PU.1 were identified under the false discovery rate 0.1% in NB4 cells. PU.1 bound to various types of genomic regions and acted as a promoter-enhancer dual binding transcription factor. Based on the sequence length and composition, two types of representative motifs were identified in PU.1 binding sites: a long and a short motif. The long motif, characterized by high sequence specificity and binding affinity, predominantly resided in the promoter-distal regions. In contrast, the short one, with strong evolutionary constraint, represented the primary PU.1 cis-elements in the promoter-proximal regions. Interestingly, the short one showed more preference to be correlated with the binding of other factors, especially PML/RARα. Moreover, genes targeted by both PU.1 and PML/RARα were significantly involved in categories associated with oncogenesis, hematopoiesis and the pathogenesis of acute myeloid leukemia.ConclusionsOur results demonstrate that structurally differentiated cis-elements that interact with PU.1 are functionally distinguishable in APL, suggesting that the sequence diversity of cis-elements might be a critical mechanism by which cells interpret the genome, and contribute to distinct physiological and/or pathological function.

Overexpressed long noncoding RNA CRNDE with distinct alternatively spliced isoforms in multiple cancers

Alternative splicing is a tightly regulated process that contributes to cancer development. CRNDE is a long noncoding RNA with alternative splicing and is implicated in the pathogenesis of several cancers. However, whether deregulated expression of CRNDE is common and which isoforms are mainly involved in cancers remain unclear. In this study, we report that CRNDE is aberrantly expressed in the majority of solid and hematopoietic malignancies. The investigation of CRNDE expression in normal samples revealed that CRNDE was expressed in a tissue- and cell-specific manner. Further comparison of CRNDE expression in 2938 patient samples from 15 solid and hematopoietic tumors showed that CRNDE was significantly overexpressed in 11 malignancies, including 3 reported and 8 unreported, and also implicated that the overexpressed isoforms differed in various cancer types. Furthermore, anti-cancer drugs could efficiently repress CRNDE overexpression in cancer cell lines and primary samples, and even had different impacts on the expression of CRNDE isoforms. Finally, experimental profiles of 12 alternatively spliced isoforms demonstrated that the spliced variant CRNDE-g was the most highly expressed isoform in multiple cancer types. Collectively, our results emphasize the cancer-associated feature of CRNDE and its spliced isoforms, and may provide promising targets for cancer diagnosis and therapy.

An organogenesis network-based comparative transcriptome analysis for understanding early human development in vivo and in vitro

BackgroundIntegrated networks hold great promise in a variety of contexts. In a recent study, we have combined expression and interaction data to identify a putative network underlying early human organogenesis that contains two modules, the stemness-relevant module (hStemModule) and the differentiation-relevant module (hDiffModule). However, owing to its hypothetical nature, it remains unclear whether this network allows for comparative transcriptome analysis to advance our understanding of early human development, both in vivo and in vitro.ResultsBased on this integrated network, we here report comparisons with the context-dependent transcriptome data from a variety of sources. By viewing the network and its two modules as gene sets and conducting gene set enrichment analysis, we demonstrate the networks utility as a quantitative monitor of the stem potential versus the differentiation potential. During early human organogenesis, the hStemModule reflects the generality of a gradual loss of the stem potential. The hDiffModule indicates the stage-specific differentiation potential and is therefore not suitable for depicting an extended developmental window. Processing of cultured cells of different types further revealed that the hStemModule is a general indicator that distinguishes different cell types in terms of their stem potential. In contrast, the hDiffModule cannot distinguish between differentiated cells of different types but is able to predict differences in the differentiation potential of pluripotent cells of different origins. We also observed a significant positive correlation between each of these two modules and early embryoid bodies (EBs), which are used as in vitro differentiation models. Despite this, the network-oriented comparisons showed considerable differences between the developing embryos and the EBs that were cultured in vitro over time to try to mimic in vivo processes.ConclusionsWe strongly recommend the use of these two modules either when pluripotent cell types of different origins are involved or when the comparisons made are constrained to the in vivo embryos during early human organogenesis (and an equivalent in vitro differentiation models). Network-based comparative transcriptome analysis will contribute to an increase in knowledge about human embryogenesis, particularly when only transcriptome data are currently available. These advances will add an extra dimension to network applications.

Repression of CDKN2C caused by PML/RARα binding promotes the proliferation and differentiation block in acute promyelocytic leukemia.

Inappropriate cell proliferation during oncogenesis is often accompanied by inactivation of components involved in the cell cycle machinery. Here, we report that cyclin-dependent kinase inhibitor 2C (CDKN2C) as a member of the cyclin-dependent kinase inhibitors is a target of the PML/RARα oncofusion protein in leukemogenesis of acute promyelocytic leukemia (APL).We found that CDKN2C was markedly downregulated in APL blasts compared with normal promyelocytes. Chromatin immunoprecipitation combined with quantitative polymerase chain reaction demonstrated that PML/RARα directly bound to the CDKN2C promoter in the APL patient-derived cell line NB4. Luciferase assays indicated that PML/RARα inhibited the CDKN2C promoter activity in a dose-dependent manner. Furthermore, all-trans retinoic acid treatment induced CDKN2C expression by releasing the PML/RARα binding on chromatin in NB4 cells. Functional studies showed that ectopic expression of CDKN2C induced a cell cycle arrest at the G0/G1 phase and a partial differentiation in NB4 cells. Finally, the transcriptional regulation of CDKN2C was validated in primary APL patient samples. Collectively, this study highlights the importance of CDKN2C inactivation in the abnormal cell cycle progression and differentiation block of APL cells and may provide new insights into the study of pathogenesis and targeted therapy of APL.