No abstract

Hematopoietic clones harboring specific mutations may expand over time. However, it remains unclear how different cellular stressors influence this expansion. Here we characterize clonal hematopoiesis after two different cellular stressors: cytotoxic therapy and hematopoietic transplantation. Cytotoxic therapy results in the expansion of clones carrying mutations in DNA damage response genes, including TP53 and PPM1D. Analyses of sorted populations show that these clones are typically multilineage and myeloid-biased. Following autologous transplantation, most clones persist with stable chimerism. However, DNMT3A mutant clones often expand, while PPM1D mutant clones often decrease in size. To assess the leukemic potential of these expanded clones, we genotyped 134 t-AML/t-MDS samples. Mutations in non-TP53 DNA damage response genes are infrequent in t-AML/t-MDS despite several being commonly identified after cytotoxic therapy. These data suggest that different hematopoietic stressors promote the expansion of distinct long-lived clones, carrying specific mutations, whose leukemic potential depends partially on the mutations they harbor.

RNA folding in the cell occurs during transcription. Expedient RNA folding must avoid the formation of undesirable structures as the nascent RNA emerges from the RNA polymerase. We show that efficient folding during transcription of three conserved noncoding RNAs from Escherichia coli, RNase P RNA, signal-recognition particle RNA, and tmRNA is facilitated by their cognate polymerase pausing at specific locations. These pause sites are located between the upstream and downstream portions of all of the native long-range helices in these noncoding RNAs. In the paused complexes, the nascent RNAs form labile structures that sequester these upstream portions in a manner to possibly guide folding. Both the pause sites and the secondary structure of the nonnative portions of the paused complexes are phylogenetically conserved among ␥-proteobacteria. We propose that specific pausing-induced structural formation is a general strategy to facilitate the folding of long-range helices. This polymerase-based mechanism may result in portions of noncoding RNA sequences being evolutionarily conserved for efficient folding during transcription.RNase P ͉ tmRNA ͉ long-range helix B ecause of the degeneracy and stability of base-pairing, RNA has a high propensity to form long-lived undesirable intermediates along their folding pathways (1-3). Particularly challenging for RNA folding during transcription is the formation of duplexes composed of two strands located far apart in sequence. In the time before the transcription of the downstream strand, the upstream strand can form stable yet nonnative base pairs with other regions, which could hinder folding. This undesirable possibility can be circumvented by sequestering the upstream strand in specific but labile structures. Such structures can guide the formation of the native long-range helices by avoiding the formation of long-lived unproductive species.When compared with Mg 2ϩ -induced refolding of full-length RNAs, two obvious properties that influence RNA folding during transcription are the 5Ј to 3Ј polarity of RNA synthesis and the transcriptional speed of the RNA polymerase (4-8). In addition, bacterial RNA polymerases pause during transcriptional elongation (9, 10). The location and/or duration of the pause sites can alter folding (7, 11). A previous work on the folding during transcription of a circularly permuted version of the Bacillus subtilis RNase P RNA demonstrated that pausing at a specific site alters its folding during transcription by Escherichia coli RNA polymerase (11). Only the pause at this site was required for the accelerated folding of one of the two domains of P RNA; varying the transcriptional speed had no effect. Pausing at this site altered the nascent RNA so that an undesirable structure involving a region downstream to the pause site was avoided.Although the above result demonstrated that pausing can influence folding, it did not address the question of biological relevancy, because (i) circularly permuted RNA transcripts are unnatural, having changed t...

Mutations in the WNT/beta-catenin pathway are responsible for initiating the majority of colorectal cancers (CRCs). We have previously shown that hyperactivation of this signaling by histone deacetylase inhibitors (HDACis) such as butyrate, a fermentation product of dietary fiber, promotes CRC cell apoptosis. The extent of association between beta-catenin and the transcriptional coactivator CREB-binding protein (CBP) influences WNT/catenin signaling and, therefore, colonic cell physiology. CBP functions as a histone acetylase (HAT); therefore, we hypothesized that the modulation of WNT/catenin activity by CBP modifies the ability of the HDACi butyrate to hyperinduce WNT signaling and apoptosis in CRC cells. Our findings indicate that CBP affects the hyperinduction of WNT activity by butyrate. ICG-001, which specifically blocks association between CBP and beta-catenin, abrogates the butyrate-triggered increase in the number of CRC cells with high levels of WNT/catenin signaling. Combination treatment of CRC cells with ICG-001 and butyrate results in cell type-specific effects on apoptosis. Further, both butyrate and ICG-001 repress CRC cell proliferation, with additive effects in suppressing cell growth. Our study strongly suggests that ICG-001-like agents would be effective against butyrate/HDACi-resistant CRC cells. Therefore, ICG-001-like agents may represent an important therapeutic option for CRCs that exhibit low-fold hyperactivation of WNT activity and apoptosis in the presence of HDACis. The findings generated from this study may lead to approaches that utilize modulation of CBP activity to facilitate CRC therapeutic or chemopreventive strategies.

Point mutations in the seed sequence of miR-142-3p are present in a subset of acute myelogenous leukemia (AML) and in several subtypes of B-cell lymphoma. Here, we show that mutations associated with AML result both in loss of miR-142-3p function and in decreased miR-142-5p expression. loss altered the hematopoietic differentiation of multipotent hematopoietic progenitors, enhancing their myeloid potential while suppressing their lymphoid potential. During hematopoietic maturation, loss of increased ASH1L protein expression and consequently resulted in the aberrant maintenance of gene expression in myeloid-committed hematopoietic progenitors. loss also enhanced the disease-initiating activity of -mutant hematopoietic cells in mice. Together these data suggest a novel model in which miR-142, through repression of ASH1L activity, plays a key role in suppressing expression during normal myeloid differentiation. AML-associated loss-of-function mutations of disrupt this negative signaling pathway, resulting in sustained expression in myeloid progenitors/myeloblasts and ultimately contributing to leukemic transformation. These findings provide mechanistic insights into the role of miRNAs in leukemogenesis and hematopoietic stem cell function. .