[Show abstract][Hide abstract] ABSTRACT: In cardiomyocytes, calcium is known to control gene expression at the level of transcription, whereas its role in regulating alternative splicing has not been explored. Here we report that, in mouse primary or embryonic stem cell-derived cardiomyocytes, increased calcium levels induce robust and reversible skipping of several alternative exons from endogenously expressed genes. Interestingly, we demonstrate a calcium-mediated splicing regulatory mechanism that depends on changes of histone modifications. Specifically, the regulation occurs through changes in calcium-responsive kinase activities that lead to alterations in histone modifications and subsequent changes in the transcriptional elongation rate and exon skipping. We demonstrate that increased intracellular calcium levels lead to histone hyperacetylation along the body of the genes containing calcium-responsive alternative exons by disrupting the histone deacetylase-to-histone acetyltransferase balance in the nucleus. Consequently, the RNA polymerase II elongation rate increases significantly on those genes, resulting in skipping of the alternative exons. These studies reveal a mechanism by which calcium-level changes in cardiomyocytes impact on the output of gene expression through altering alternative pre-mRNA splicing patterns.
Full-text · Article · Nov 2014 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Positron emission tomography (PET) imaging using 2-deoxy-2-[F-18]fluoro-D-glucose (FDG) has proven valuable in the diagnosis, staging and restaging for many cancers. However, its application for liver cancer has remained limited owing in part to the relatively high background uptake of the tracer in the liver plus the significant variability of the tumor specific uptake in liver cancer among patients. Thus, for primarily liver cancer, in particular, hepatocellular carcinoma (HCC), radio-tracers with better tumor-enhancing uptake/retention are still sought in order to harness the great power of PET imaging. Here, we reviewed some recent investigations with lipid-based small molecule PET radio-tracers with relevance to fasting, and discuss their potential in the diagnosis and staging of HCCs.
[Show abstract][Hide abstract] ABSTRACT: Neurofibromatosis type I (Nf1) is a GTPase-activating protein (GAP) that inactivates the oncoprotein Ras and plays important
roles in nervous system development and learning. Alternative exon 23a falls within the Nf1 GAP domain coding sequence and is tightly regulated in favor of skipping in neurons; however, its biological function is
not fully understood. Here we generated mouse embryonic stem (ES) cells with a constitutive endogenous Nf1 exon 23a inclusion, termed Nf1 23aIN/23aIN cells, by mutating the splicing signals surrounding the exon to better match consensus sequences. We also made Nf1 23aΔ/23aΔ cells lacking the exon. Active Ras levels are high in wild-type (WT) and Nf1 23aIN/23aIN ES cells, where the Nf1 exon 23a inclusion level is high, and low in Nf1 23aΔ/23aΔ cells. Upon neuronal differentiation, active Ras levels are high in Nf1 23aIN/23aIN cells, where the exon inclusion level remains high, but Ras activation is low in the other two genotypes, where the exon
is skipped. Signaling downstream of Ras is significantly elevated in Nf1 23aIN/23aIN neurons. These results suggest that exon 23a suppresses the Ras-GAP activity of Nf1. Therefore, regulation of Nf1 exon 23a inclusion serves as a mechanism for providing appropriate levels of Ras signaling and may be important in modulating
Ras-related neuronal functions.
[Show abstract][Hide abstract] ABSTRACT: R-spondins are a family of secreted Wnt agonists. One of the family members, R-spondin 2 (RSPO2), has an important role in embryonic development, bone formation and myogenic differentiation; however, its role in human cancers remains largely unknown. Here we show that RSPO2 expression is downregulated in human colorectal cancers (CRCs) due to promoter hypermethylation, and that the RSPO2 reduction correlates with tumour differentiation, size and metastasis. Overexpression of RSPO2 suppresses CRC cell proliferation and tumorigenicity, whereas the depletion of RSPO2 enhances tumour cell growth. RSPO2 has an inhibitory effect on Wnt/β-catenin signaling in the CRC cells that show suppressed cell proliferation. In human CRC cells, the RSPO2-induced inhibition of Wnt signaling depends on leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5); RSPO2 interacts with LGR5 to stabilize the membrane-associated zinc and ring finger 3 (ZNRF3). Our data suggest that RSPO2 functions as a tumour suppressor in human CRCs, and these data reveal a RSPO2-induced, LGR5-dependent Wnt signaling-negative feedback loop that exerts a net growth-suppressive effect on CRC cells.
No preview · Article · Jan 2014 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: The Protein Kinase Inhibitor (Pki) gene family inactivates nuclear PKA and terminates PKA-induced gene expression. We previously showed that Pkig is the primary family member expressed in osteoblasts and that Pkig knockdown increases the effects of parathyroid hormone and isoproterenol on PKA activation, gene expression, and inhibition of apoptosis. Here, we determined whether endogenous levels of Pkig regulate osteoblast differentiation. Pkig is the primary family member in MEFs, murine marrow-derived mesenchymal stem cells, and human mesenchymal stem cells. Pkig deletion increased forskolin-dependent nuclear PKA activation and gene expression and Pkig deletion or knockdown increased osteoblast differentiation. PKA signaling is known to stimulate adipogenesis; however, adipogenesis and osteogenesis are often reciprocally regulated. We found that the reciprocal regulation predominates over the direct effects of PKA since adipogenesis was decreased by Pkig deletion or knockdown. Pkig deletion or knockdown simultaneously increased osteogenesis and decreased adipogenesis in mixed osteogenic/adipogenic medium. Pkig deletion increased PKA-induced expression of Leukemia Inhibitory Factor (Lif) mRNA and LIF protein. LIF neutralizing antibodies inhibited the effects on osteogenesis and adipogenesis of either Pkig deletion in MEFs or PKIγ knockdown in both murine and human mesenchymal stem cells. Collectively, our results show that endogenous levels of Pkig reciprocally regulate osteoblast and adipocyte differentiation and that this reciprocal regulation is mediated in part by LIF. Stem Cells 2013.
[Show abstract][Hide abstract] ABSTRACT: The molecular mechanisms through which alternative splicing and histone modifications regulate gene expression are now understood in considerable detail. Here, we discuss recent studies that connect these two previously separate avenues of investigation, beginning with the unexpected discoveries that nucleosomes are preferentially positioned over exons and DNA methylation and certain histone modifications also show exonic enrichment. These findings have profound implications linking chromatin structure, histone modification and splicing regulation. Complementary single gene studies provided insight into the mechanisms through which DNA methylation and histones modifications modulate alternative splicing patterns. Here, we review an emerging theme resulting from these studies: RNA-guided mechanisms integrating chromatin modification and splicing. Several groundbreaking papers reported that small noncoding RNAs affect alternative exon usage by targeting histone methyltransferase complexes to form localized facultative heterochromatin. More recent studies provided evidence that pre-messenger RNA itself can serve as a guide to enable precise alternative splicing regulation via local recruitment of histone-modifying enzymes, and emerging evidence points to a similar role for long noncoding RNAs. An exciting challenge for the future is to understand the impact of local modulation of transcription elongation rates on the dynamic interplay between histone modifications, alternative splicing and other processes occurring on chromatin.
Full-text · Article · Sep 2013 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: The assembly and disassembly of ribonucleoproteins (RNPs) are dynamic processes that control every step of RNA metabolism, including mRNA stability. However, our knowledge of how RNP remodeling is achieved is largely limited to RNA helicase functions. Here, we report a previously unknown mechanism that implicates the ATPase p97, a protein-remodeling machine, in the dynamic regulation of mRNP disassembly. We found that p97 and its cofactor, UBXD8, destabilize p21, MKP-1, and SIRT1, three established mRNA targets of the RNA-binding protein HuR, by promoting release of HuR from mRNA. Importantly, ubiquitination of HuR with a short K29 chain serves as the signal for release. When cells are subjected to stress conditions, the steady-state levels of HuR ubiquitination change, suggesting a new mechanism through which HuR mediates the stress response. Our studies reveal a new paradigm in RNA biology: nondegradative ubiquitin signaling-dependent disassembly of mRNP promoted by the p97-UBXD8 complex to control mRNA stability.
Full-text · Article · Apr 2013 · Genes & development
[Show abstract][Hide abstract] ABSTRACT: A group of radiolabeled thymidine analogs were developed as radio-tracers for imaging herpes viral thymidine kinase (HSV1-tk) or its variants used as reporter gene. A transgenic mouse model was created to express tk upon liver injury or naturally occurring hepatocellular carcinoma (HCC). The purpose of this study was to use this unique animal model for initial testing with radio-labeled thymidine analogs, mainly a pair of newly emerging nucleoside analogs, D-FMAU and L-FMAU.
A transgeneic mouse model was created by putting a fused reporter gene system, firefly luciferase (luc) and HSV1-tk, under the control of mouse alpha fetoprotein (Afp) promoter. Initial multimodal imaging, which was consisted of bioluminescent imaging (BLI) and planar gamma scintigraphy with [(125)I]-FIAU, was used for examining the model creation in the new born and liver injury in the adult mice. Carcinogen diethylnitrosamine (DEN) was then administrated to induce HCC in these knock-in mice such that microPET imaging could be used to track the activity of Afp promoter during tumor development and progression by imaging tk expression first with [(18)F]-FHBG. Dynamic PET scans with D-[(18)F]-FMAU and L-[(18)F]-FMAU were then performed to evaluate this pair of relatively new tracers. Cells were derived from these liver tumors for uptake assays using H-3 labeled version of PET tracers.
The mouse model with dual reporters: HSV1-tk and luc placed under the transcriptional control of an endogenous Afp promoter was used for imaging studies. The expression of the Afp gene was highly specific in proliferative hepatocytes, in regenerative liver, and in developing fetal liver, and thus provided an excellent indicator for liver injury and cancer development in adult mice. Both D-FMAU and L-FMAU showed stable liver tumor uptake where the tk gene was expressed under the Afp promoter. The performance of this pair of tracers was slightly different in terms of signal-to-background ratio as well as tracer clearance.
The newly created knock-in mouse model was used to demonstrate the use of the dual-reporter genes driven by well-characterized cancer-specific transcriptional units in conjunction with in vivo imaging as a paradigm in studying naturally occurring cancer in live animals. While BLI is suitable for small animal imaging with luc expression, PET with L-FMAU seemed be the choice for liver injury or liver cancer imaging with this animal model for future investigations.
[Show abstract][Hide abstract] ABSTRACT: Camptothecins (CPTs) are an important class of effective anticancer agents that target type I topoisomerase in humans. Irinotecan and topotecan are currently used to treat various types of cancers and many CPT derivatives are being developed. However, these drugs are only effective in a small percentage of each type of cancers and the molecular underpinning for this individualized response to the drug has remained elusive. Thus, identification of the main determinants for cell survival in response to this unique class of drug should help to improve their clinical applications. In this study, we examined whether RECQL5 constituted an important determinant of CPT resistance in colon cancer cells. Specifically, RECQL5 deficient derivatives of both DDL1 and HCT116 cells, two colorectal cancer cell lines were generated by adenovirus-based somatic gene targeting experiments and the CPT sensitivity between the RECQL5 proficient parental lines and their corresponding RECQL5 deficient derivatives were examined. We found that deletion of RECQL5 from DDL1 and HCT116 both resulted in a significant enhancement in CPT sensitivity under in vitro cultured condition. More importantly, xenograft tumors derived from RECQL5 deficient HCT116 cells but not those from the parental line could be cured by a CPT-based therapy in nude mice. Thus, this study has identified RECQL5 as a major determinant for CPT resistance in colorectal cancer cells and a potential candidate as a biomarker for irinotecan-based treatment for colon cancer.
Full-text · Article · Oct 2011 · Bioscience Reports
[Show abstract][Hide abstract] ABSTRACT: Recent studies have provided strong evidence for a regulatory link among chromatin structure, histone modification, and splicing regulation. However, it is largely unknown how local histone modification patterns surrounding alternative exons are connected to differential alternative splicing outcomes. Here we show that splicing regulator Hu proteins can induce local histone hyperacetylation by association with their target sequences on the pre-mRNA surrounding alternative exons of two different genes. In both primary and mouse embryonic stem cell-derived neurons, histone hyperacetylation leads to an increased local transcriptional elongation rate and decreased inclusion of these exons. Furthermore, we demonstrate that Hu proteins interact with histone deacetylase 2 and inhibit its deacetylation activity. We propose that splicing regulators may actively modulate chromatin structure when recruited to their target RNA sequences cotranscriptionally. This "reaching back" interaction with chromatin provides a means to ensure accurate and efficient regulation of alternative splicing.
Full-text · Article · Aug 2011 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Deficiencies in DNA damage response and repair not only can result in genome instability and cancer predisposition, but also
can render the cancer cells intrinsically more vulnerable to certain types of DNA damage insults. Particularly, replication
stress is both a hallmark of human cancers and a common instigator for genome instability and cell death. Here, we review
our work based on the genetic knockout studies on Blm and Recql5, two members of the mammalian RecQ helicase family. These studies have uncovered a unique partnership between these two helicases in the implementation of proper
mitigation strategies under different circumstances to promote DNA replication and cell survival and suppress genome instability
and cancer. In particular, current studies have revealed the presence of a novel Recql5/RECQL5-dependent mechanism for suppressing
replication fork collapse in response to global replication fork stalling following exposure to camptothecin (CPT), a topoisomerase
I inhibitor, and a potent inhibitor of DNA replication. The unique partnership between Blm and Recql5 in coping with the challenge
imposed by replication stress is discussed. In addition, given that irinotecan and topotecan, two CPT derivatives, are currently
used in clinic for treating human cancer patients with very promising results, the potential implication of the new findings
from these studies in anticancer treatments is also discussed.
Full-text · Article · Feb 2011 · Journal of Molecular Cell Biology
[Show abstract][Hide abstract] ABSTRACT: Hepatocellular carcinoma (HCC) is frequently a lethal disease and one of the few malignancies that is still increasing in incidence around the world. Better animal models are highly desired to investigate the molecular basis of HCC and to develop novel therapeutic strategies. Alpha-fetoprotein (Afp) gene is expressed in fetal liver, silenced soon after birth, and highly re-expressed in hepatocellular carcinomas (HCC). We aimed to take advantage of the dramatic re-expression of the Afp gene in HCC to develop a hepatocarcinogenesis reporter (HCR) mouse model for dual-modality, longitudinal in vivo imaging of liver tumor development, and progression.
Knock in mice were established by placing a thymidinekinase (tk)-luciferase (luc) reporter gene cassette under the transcriptional control of the endogenous Afp promoter. DEN, a liver carcinogen, was used to induce liver tumors, which was monitored by both luc-based bioluminescent (BL) and tk-based positron emission tomography (PET) imaging.
The expression profile of luc was identical to that of the endogenous Afp gene during development. As early as 2 months after the exposure to DEN, BLI revealed multifocal signals in the liver, long before the appearance of histologically apparent neoplastic lesions. By 6 months, BL and PET dual imaging showed strong signals in malignant HCC. By serendipity, a strong BL signal was also detected in adult testes, a previously unknown site of Afp expression.
The HCR model enables longitudinal monitoring of liver tumor development and progression, providing a powerful tool in developing chemoprevention and therapeutic strategies for HCC.
Full-text · Article · Dec 2010 · Journal of Hepatology
[Show abstract][Hide abstract] ABSTRACT: To investigate whether Recql5, a DNA helicase that plays an important role in the maintenance of genome integrity, is a tumor suppressor in the gastrointestinal tract in mice.
We generated cohorts of both Recql5-proficient and Recql5-deficient Apc(min/+) mice and compared the tumor susceptibility in their gastrointestinal tracts.
Recql5 deficiency in Apc(min/+) mice resulted in a significant increase in the tumor incidence in both the colon (P = 0.0162) and the small intestine (P < 0.01). These findings have provided the first genetic evidence for a tumor suppression role of Recql5 in the gastrointestinal tract of mice. Importantly, since mouse Recql5 and human RECQL5 are highly conserved, these findings also suggest that RECQL5 may be a tumor suppressor for human colon cancer.
Recql5 has a tumor suppression role in the mouse gastrointestinal tract.
Full-text · Article · Mar 2010 · World Journal of Gastroenterology
[Show abstract][Hide abstract] ABSTRACT: RecQ helicases are members of an evolutionary conserved family of DNA helicases. They are homologous to the RecQ helicase of E. coli, the founding member of the family. These enzymes include gene products of disease-causing genes in Bloom, Werner, and Rothmund-Thomson syndrome. To date, these proteins have been implicated in many aspects of DNA metabolism, including DNA replication, repair, and recombination. We reported here that RECQL5, a newer member of the human RecQ helicase family, physically interacts with SWI/SNF complex and RNAPII core complex within the context of a super complex. RECQL5 was detected in the RNAPII holoenzyme but not in purified RNAPII core complex. Together, these data link RECQL5 to the assembly of the RNAPII transcription machinery and suggest that this helicase may have a regulatory role in RNAPII transcription or an RNAPII-related process or processes.
Full-text · Article · Jan 2010 · International Journal of Biochemistry and Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: A new spontaneous mouse mutant (ntl) with autosomal-recessive osteopetrosis was characterized. These mice formed tartrate-resistant acid phosphate (TRAP)-positive osteoclasts but their osteoclasts had no ruffled border and did not resorb bone. These mice displayed no tooth eruption or tooth root formation. Adult mutant mice developed odontoma-like proliferations near the proximal ends of the incisors. Intraperitoneal injection of progenitor cells from the liver of 16.5 days postcoitum wild-type embryos into newborn mutants rescued the osteopetrosis phenotype, indicating that the defects were intrinsic to the osteoclasts. Our findings not only provide further support for a critical role of osteoclasts in tooth eruption and tooth root development, but also suggest that the perturbation of the homeostasis of the odontogenic precursors of the incisors is primarily responsible for the development of the odontoma-like proliferations in this osteopetrosis mutant. Genetic mapping has narrowed down the location of the mutant allele to a genetic interval of 3.2 cM on mouse chromosome 17.
No preview · Article · Dec 2009 · European Journal Of Oral Sciences
[Show abstract][Hide abstract] ABSTRACT: Disruption of replication can lead to loss of genome integrity and increase of cancer susceptibility in mammals. Thus, a replication impediment constitutes a formidable challenge to these organisms. Recent studies indicate that homologous recombination (HR) plays an important role in suppressing genome instability and promoting cell survival after exposure to various replication inhibitors, including a topoisomerase I inhibitor, camptothecin (CPT). Here, we report that the deletion of RecQ helicase Recql5 in mouse ES cells and embryonic fibroblast (MEF) cells resulted in a significant increase in CPT sensitivity and a profound reduction in DNA replication after the treatment with CPT, but not other DNA-damaging agents. This CPT-induced cell death is replication dependent and occurs primarily after the cells had exited the first cell cycle after CPT treatment. Furthermore, we show that Recql5 functions nonredundantly with Rad51, a key factor for HR to protect mouse ES cells from CPT-induced cytotoxicity. These new findings strongly suggest that Recql5 plays an important role in maintaining active DNA replication to prevent the collapse of replication forks and the accumulation of DSBs in order to preserve genome integrity and to prevent cell death after replication stress as a result of topoisomerase I poisoning.
Full-text · Article · Dec 2008 · Molecular biology of the cell
[Show abstract][Hide abstract] ABSTRACT: High-molecular-weight kininogen (HK) plays an important role in the assembly of the plasma kallikrein-kinin system. While the human genome contains a single copy of the kininogen gene, 3 copies exist in the rat (1 encoding K-kininogen and 2 encoding T-kininogen). Here, we confirm that the mouse genome contains 2 homologous kininogen genes, mKng1 and mKng2, and demonstrate that these genes are expressed in a tissue-specific manner. To determine the roles of these genes in murine development and physiology, we disrupted mKng1, which is expressed primarily in the liver. mKng1(-/-) mice were viable, but lacked plasma HK and low-molecular-weight kininogen (LK), as well as DeltamHK-D5, a novel kininogen isoform that lacks kininogen domain 5. Moreover, despite normal tail vein bleeding times, mKng1(-/-) mice displayed a significantly prolonged time to carotid artery occlusion following Rose Bengal administration and laser-induced arterial injury. These results suggest that a single gene, mKng1, is responsible for production of plasma kininogen, and that plasma HK contributes to induced arterial thrombosis in mice.
[Show abstract][Hide abstract] ABSTRACT: Members of the RecQ helicase family play critical roles in genome maintenance. There are five RecQ homologs in mammals, and defects in three of these (BLM, WRN, and RECQL4) give rise to cancer predisposition syndromes in humans. RECQL and RECQL5 have not been associated with a human disease. Here we show that deletion of Recql5 in mice results in cancer susceptibility. Recql5-deficient cells exhibit elevated frequencies of spontaneous DNA double-strand breaks and homologous recombination (HR) as scored using a reporter that harbors a direct repeat, and are prone to gross chromosomal rearrangements in response to replication stress. To understand how RECQL5 regulates HR, we use purified proteins to demonstrate that human RECQL5 binds the Rad51 recombinase and inhibits Rad51-mediated D-loop formation. By biochemical means and electron microscopy, we show that RECQL5 displaces Rad51 from single-stranded DNA (ssDNA) in a reaction that requires ATP hydrolysis and RPA. Together, our results identify RECQL5 as an important tumor suppressor that may act by preventing inappropriate HR events via Rad51 presynaptic filament disruption.
Full-text · Article · Jan 2008 · Genes & Development
[Show abstract][Hide abstract] ABSTRACT: Recent advances in genome-wide analysis of alternative splicing indicate that extensive alternative RNA processing is associated with many proteins that play important roles in the nervous system. Although differential splicing and polyadenylation make significant contributions to the complexity of the nervous system, our understanding of the regulatory mechanisms underlying the neuron-specific pathways is very limited. Mammalian neuron-specific embryonic lethal abnormal visual-like Hu proteins (HuB, HuC, and HuD) are a family of RNA-binding proteins implicated in neuronal differentiation and maintenance. It has been established that Hu proteins increase expression of proteins associated with neuronal function by up-regulating mRNA stability and/or translation in the cytoplasm. We report here a novel function of these proteins as RNA processing regulators in the nucleus. We further elucidate the underlying mechanism of this regulation. We show that in neuron-like cells, Hu proteins block the activity of TIA-1/TIAR, two previously identified, ubiquitously expressed proteins that promote the nonneuronal pathway of calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA processing. These studies define not only the first neuron-specific regulator of the calcitonin/CGRP system but also the first nuclear function of Hu proteins.
Full-text · Article · Jan 2007 · Molecular Biology of the Cell
[Show abstract][Hide abstract] ABSTRACT: 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is a prostaglandin-degrading enzyme that is highly expressed in normal colon mucosa but is ubiquitously lost in human colon cancers. Herein, we demonstrate that 15-PGDH is active in vivo as a highly potent suppressor of colon neoplasia development and acts in the colon as a required physiologic antagonist of the prostaglandin-synthesizing activity of the cyclooxygenase 2 (COX-2) oncogene. We first show that 15-PGDH gene knockout induces a marked 7.6-fold increase in colon tumors arising in the Min (multiple intestinal neoplasia) mouse model. Furthermore, 15-PGDH gene knockout abrogates the normal resistance of C57BL/6J mice to colon tumor induction by the carcinogen azoxymethane (AOM), conferring susceptibility to AOM-induced adenomas and carcinomas in situ. Susceptibility to AOM-induced tumorigenesis is mediated by a marked induction of dysplasia, proliferation, and cyclin D1 expression throughout microscopic aberrant crypt foci arising in 15-PGDH null colons and is concomitant with a doubling of prostaglandin E(2) in 15-PGDH null colonic mucosa. A parallel role for 15-PGDH loss in promoting the earliest steps of colon neoplasia in humans is supported by our finding of a universal loss of 15-PGDH expression in microscopic colon adenomas recovered from patients with familial adenomatous polyposis, including adenomas as small as a single crypt. These models thus delineate the in vivo significance of 15-PGDH-mediated negative regulation of the COX-2 pathway and moreover reveal the particular importance of 15-PGDH in opposing the neoplastic progression of colonic aberrant crypt foci.
Full-text · Article · Sep 2006 · Proceedings of the National Academy of Sciences