Trithorax group proteins: switching genes on and keeping them active

Cellular memory is provided by two counteracting groups of chromatin proteins termed Trithorax group (TrxG) and Polycomb group (PcG) proteins. TrxG proteins activate transcription and are perhaps best known because of the involvement of the TrxG protein MLL in leukaemia. However, in terms of molecular analysis, they have lived in the shadow of their more famous counterparts, the PcG proteins. Recent advances have improved our understanding of TrxG protein function and demonstrated that the heterogeneous group of TrxG proteins is of critical importance in the epigenetic regulation of the cell cycle, senescence, DNA damage and stem cell biology

The Drosophila retained/dead ringer gene and ARID gene family function during development

© UBC PressThe recently discovered ARID family of proteins interact with DNA through a phylogenetically conserved sequence termed the A/T Interaction Domain (ARID). The retained/dead ringer (retn/dri) gene of Drosophila melanogaster is a founding member of the ARID gene family, and of the eARID subfamily. This subfamily exhibits an extended region of sequence similarity beyond the core ARID motif and a separate conserved domain termed the REKLES domain. retn/dri is involved in a range of developmental processes, including axis patterning and muscle development. The retn/dri ARID motif has been shown by in vitro studies to exhibit sequence-specific DNA binding activity. Here we demonstrate that the ARID domain is essential for the in vivo function of retn/dri during embryonic development by showing that a mutant form of RETN/DRI, deleted for part of the ARID domain and unable to bind DNA in vitro, cannot rescue the retn/dri mutant phenotype. In the presence of wild-type RETN/DRI this construct acts as a dominant negative, providing additional support for the proposal that RETN/DRI acts in a multiprotein complex. In contrast, we are yet to find an in vivo role for the REKLES domain, despite its clear evolutionary conservation. Finally, we have used germline clone analysis to reveal a requirement for retn/dri in the Drosophila preblastoderm syncytial mitoses.Tetyana Shandala, R. Daniel Kortschak and Robert Sain

miR-335-Oct4-Rb1: a new axis to control cancer cell proliferation and self-renewal

2011/2012The retinoblastoma tumor suppressor protein (pRb) belongs to a cellular pathway that is deregulated in several human tumors. A hallmark of the pRb pathway is its ability to control G1-S transition of the cell cycle and to prevent uncontrolled cell proliferation. Nevertheless, research in the last years identified multiple alternative cellular functions regulated by pRb including several aspects of stem cell biology, that in contrast to pRb role in cell cycle control, are poorly understood. The work presented in this thesis aimed to investigate the post-transcriptional regulation of pRb expression and pRb function in the context of human cancer and stem cell biology. In the first part of this thesis work we evaluated the role of miR-335, a microRNA predicted to target RB1, to regulate pRb at post-transcriptional level and the impact of this regulation on pRb tumor suppressor function in cancer cells, focusing on the control of cell proliferation; in the second part we investigate the role of pRb in stem cells context studying the relationship between the control of cell-cycle progression and the control of self-renewal features. In particular we focused our research both on the regulation of its expression by evaluating the impact of miR-335 on pRb in the context of embryonic stem cells, and to link pluripotency transcription factor Oct4 with the regulation of the pRb pathway. MicroRNAs are small non-coding RNAs critically involved in the post-transcriptional regulation of gene expression and influencing all biological processes, including tumorigenesis, stem cell self-renewal and differentiation. Although Rb proteins have a critical role in many cellular processes, information on microRNAs that control Rb family proteins at post-transcriptional levels are still very limited. The initial aim of this project was to address a putative role for miR-335, a microRNA predicted to target mammalian RB1, in the control of pRb expression, cell proliferation and tumorigenesis. We were able to demonstrate that miR-335 is differentially expressed in human cancer cells and that it regulates the expression of human pRb by directly target a conserved sequence motif in its 3’UTR. High conservation of miR-335 and its RB1 target sequence in placental mammals, as well as differential expression in human cancer cell lines indicates an important role in cell cycle control. We found that the impairment of the pRb pathway by miR-335 is paralleled by a significant upregulation of p53 levels and that the activation of the p53 pathway in the context of miR-335 overexpression impairs cell proliferation and neoplastic transformation. In line with this, reducing p53 levels is sufficient to drive hyperproliferation and increased transformation in the context of ectopically increased miR-335 levels. In our study, we also showed that the activation of the p53 pathway during DNA damage significantly increases miR-335 levels and demonstrated that miR-335 mediates efficient cell cycle arrest on DNA damage in a positive feedback loop with p53. In conclusion, these results identify miR-335 as a potent regulator of pRb at posttranscriptional level, indicate that miR-335 helps control proliferation by balancing the activities of the pRb and p53 tumor suppressor pathway and that miR- 335 activation plays an important role in the induction of p53-dependent cell cycle arrest after DNA damage. Recently it has becoming evident that cancer cells exhibit several traits that are also characteristic of embryonic stem cells. In particular, the constitutive hyperphosphorylation of pRb that ensures rapid and indefinite cell proliferation potential of ESCs recapitulates the impaired function of the pRb pathway present in virtually all tumors. In addition, self-renewal expression signature including the pluripotency transcription factors Oct4, Nanog and Sox2 were also found in several types of human cancer. Although these evidences support the idea that the acquisition of stem cell-like gene expression signature represents a key step in tumorigenesis, evidence for a functional link between pluripotency transcription modules and the control of aberrant function of tumor suppressor pathways is limited. The second aim of this thesis work was to better characterize the role of pRb in stem cells context focusing on the regulation of its expression and function, connecting pluripotency transcription factor Oct4 with the Retinoblastoma tumor suppressor pathway. We were able to demonstrate that Oct4 cooperates with pRb in the establishment of self-renewal program of mouse embryonic stem cells (mESCs) and that these two factors are under control of miR-335 that targets conserved sequence motifs in the 3’UTRs of Oct4 and Rb1. We demonstrated that Oct4 is required to ensure the hyperphosphorylation of pRb, thereby permitting rapid cell proliferation of self-renewing mESCs. miR-335 plays a central role in interrupting the self-renewal promoting Oct4-pRb axis at the onset of mESC differentiation by targeting the expression of both proteins at the post- transcriptional level. In particular, we found that in self-renewing mESCs, high Oct4 expression levels drive the expression of Nipp1 and Ccnf to inhibit the activity of the protein phosphatase type 1 (Pp1) thereby establishing pRb hyperphosphorylation as a key feature of rapidly cycling, pluripotent mESCs. Upon induction of differentiation, transcriptional repression of Oct4 in conjunction with targeting of Rb1 and Oct4 by miR-335 causes the collapse of the Oct4-Nipp1/Ccnf1-Pp1-pRb axis, leading to a rapid pRb dephosphorylation, the exit from self-renewal and the establishment of pRb regulated cell cycle profile of differentiated cells. In conclusion, our results introduce a novel regulatory circuit, modulated by miR- 335, which connects the pluripotency transcription factor Oct4 with the pRb pathway to control mESC self-renewal and differentiation, and anticipate an important role for Oct4 in the inactivation of the Retinoblastoma tumor suppressor pathway in human cancers with embryonic stem cell gene expression signatures.XXV Ciclo198

The Nuclear Matrix Protein, NRP/B, Acts as a Transcriptional Repressor of E2F-mediated Transcriptional Activity

Background: NRP/B, a family member of the BTB/Kelch repeat proteins, is implicated in neuronal and cancer development, as well as the regulation of oxidative stress responses in breast and brain cancer. Our previous studies indicate that the NRP/B-BTB/POZ domain is involved in the dimerization of NRP/B and in a complex formation with the tumor suppressor, retinoblastoma protein. Although much evidence supports the potential role of NRP/B as a tumor suppressor, the molecular mechanisms of NRP/B action on E2F transcription factors have not been elucidated. Methods: Three-dimensional modeling of NRP/B was used to generate point mutations in the BTB/Kelch domains. Tet-on inducible NRP/B expression was established. The NRP/B deficient breast cancer cell line, MDA-MB-231, was generated using lentiviral shNRP/B to evaluate the effect of NRP/B on cell proliferation, invasion and migration. Immunoprecipitation was performed to verify the interaction of NRP/B with E2F and histone deacetylase (HDAC-1), and the expression level of NRP/B protein was analyzed by Western blot analysis. Changes in cell cycle were determined by flow cytometry. Transcriptional activities of E2F transcription factors were measured by chloramphenicol acetyltransferase (CAT) activity. Results: Ectopic overexpression of NRP/B demonstrated that the NRP/B-BTB/POZ domain plays a critical role in E2F-mediated transcriptional activity. Point mutations within the BTB/POZ domain restored E2-promoter activity inhibited by NRP/B. Loss of NRP/B enhanced the proliferation and migration of breast cancer cells. Endogenous NRP/B interacted with E2F and HDAC1. Treatement with an HDAC inhibitor, trichostatin A (TSA), abolished the NRP/B-mediated suppression of E2-promoter activity. Gain or loss of NRP/B in HeLa cells confirmed the transcriptional repressive capability of NRP/B on the E2F target genes, Cyclin E and HsORC (Homo sapiens Origin Recognition Complex). Conclusions: The present study shows that NRP/B acts as a transcriptional repressor by interacting with the co-repressors, HDAC1, providing new insight into the molecular mechanisms of NRP/B on tumor suppression

Cell cycle regulation by long non-coding RNAs

The mammalian cell cycle is precisely controlled by cyclin-dependent kinases (CDKs) and related pathways such as the RB and p53 pathways. Recent research on long non-coding RNAs (lncRNAs) indicates that many lncRNAs are involved in the regulation of critical cell cycle regulators such as the cyclins, CDKs, CDK inhibitors, pRB, and p53. These lncRNAs act as epigenetic regulators, transcription factor regulators, post-transcription regulators, and protein scaffolds. These cell cycle-regulated lncRNAs mainly control cellular levels of cell cycle regulators via various mechanisms, and may provide diversity and reliability to the general cell cycle. Interestingly, several lncRNAs are induced by DNA damage and participate in cell cycle arrest or induction of apoptosis as DNA damage responses. Therefore, deregulations of these cell cycle regulatory lncRNAs may be involved in tumorigenesis, and they are novel candidate molecular targets for cancer therapy and diagnosis

Cell cycle regulation by the B-Myb transcription factor

The expression of genes required for progression through the cell cycle is highly modulated through a regulatory axis containing the E2F transcription factor and retinoblastoma tumour suppressor protein families. One of the genes regulated through this mechanism encodes the B-Myb transcription factor, which has been shown to be critically required for early embryonal development in the mouse. Transcriptional activity of B-Myb is substantially enhanced in S phase through modification by cyclin A/cdk2, and the evidence points squarely to the major role being played by B-Myb during this phase of the cell cycle. We discuss in this review recent findings suggesting that B-Myb is a multifunctional protein that has, in addition to its transcriptional properties, the ability to interact directly with other regulators of the cell cycl

Role of cell cycle regulators in development of the inner ear

The inner ear originates from an ectodermal thickening called the otic placode. The otic placode invaginates and closes to an otic vesicle, the otocyst. The otocyst epithelium undergoes morphogenetic changes and cell differentiation, leading to the formation of the labyrinth-like mature inner ear. Epithelial-mesenchymal interactions control inner ear morphogenesis, but the modes and molecules are largely unresolved. The expressions of negative cell cycle regulators in the epithelium of the early-developing inner ear have also not been elucidated. The mature inner ear comprises the hearing (cochlea) and balance (vestibular) organs that contain the nonsensory and sensory cells. In mammals, the inner ear sensory cells, called hair cells, exit the cell cycle during embryogenesis and are mitotically quiescent during late-embryonic differentiation stages and postnatally. The mechanisms that maintain this hair cell quiescense are largely unresolved. In this work I examined 1) the epithelial-mesenchymal interactions involved in inner ear morphogenesis, 2) expression of negative cell cycle regulators in the epithelium of the early developing inner ear and 3) the molecular mechanisms that maintain the postmitotic state of inner ear sensory cells. We observed that during otocyst stages, epithelial fibroblast growth factor 9 (Fgf9) communicates with the surrounding mesenchyme, where its receptors are expressed. Fgf9 inactivation leads to reduced proliferation of the surrounding vestibular mesenchyme and to the absence of semicircular canals. Semicircular canal development is blocked, since fusion plates do not form. These results show that the mesenchyme directs fusion plate formation and give direct evidence for the existence of reciprocal epithelial-mesenchymal interactions in the developing inner ear. Cyclin-dependent kinase inhibitors (CKIs) are negative regulators of proliferation. We show that the members of the Cip/Kip family of CKIs (p21Cip1, p27Kip1 and p57Kip2) are expressed in the early-developing inner ear. Our expression data suggest that CKIs divide the otic epithelium into proliferative and nonproliferative compartments that may underlie shaping of the otocyst. At later stages, CKIs regulate proliferation of the vestibular appendages, and this may regulate their continual growth. In addition to restricting proliferation, CKIs may play a role in regional differentiation of various epithelial cells. Differentiating and adult inner ear hair cells are postmitotic and do not proliferate in response to serum or mitogenic growth factors. In our study, we show that this is the result of the activity of negative cell cycle regulators. Based on expression profiles, we first focused on the retinoblastoma (Rb) gene, which functions downstream of the CKIs. Analysis of the inner ear phenotype of Rb mutant mice show, that the retinoblastoma protein regulates the postmitotic state of hair cells. Rb inactivation leads to hyperplasia of vestibular and cochlear sensory epithelia that is a result of abnormal cell cycle entry of differentiated hair cells and of delayed cell cycle exit of the hair cell precursor cells. In addition, we show that p21Cip1 and p19Ink4d cooperate in maintaining the postmitotic state of postnatal auditory hair cells. Whereas inactivation of p19Ink4d alone leads to low-level S-phase entry (Chen et al., 2003) and p21Cip1 null mutant mice have a normal inner ear phenotype, codeletion of p19Ink4d and p21Cip1 triggers high-level S-phase entry of auditory hair cells during early postnatal life, which leads to supernumerary hair cells. The ectopic hair cells undergo apoptosis in all of the mutant mice studied, DNA damage being the immediate cause of this death. These findings demonstrate that the maintenance of the postmitotic state of hair cells is regulated by Rb and several CKIs, and that these cell cycle regulators are critical for the lifelong survival of hair cells. These data have implications for the future design of therapies to induce hair cell regrowth.Sisäkorvan kehitys alkaa ektodermin paksuuntumasta, joka kuroutuu muodostaen korvarakkulan. Sisäkorva kuulo- ja tasapainoelimineen kehittyy korvarakkulan epiteelin ja sitä ympäröivän kudoksen, mesenkyymin, vuoropuhelun seurauksena. Mekanismit ja molekyylit tämän vuoropuhelun takana ovat kuitenkin olleet epäselviä. Sisäkorvan kuulo- ja tasapainoelimissä sijaitsevat aistinsolut (karvasolut). Linnuilla ja kaloilla luonnollista karvasolujen uusiutumista tapahtuu koko eliniän aikana ja traumojen jälkeen uusiutumisprosessi kiihtyy. Nisäkkäillä uusia karvasoluja ei synny sikiövaiheen terminaalimitoosien jälkeen. Tästä syystä karvasolujen tuhoutuminen esim. melun, tiettyjen sairauksien tai ototoksisten antibioottien seurauksena on pysyvää. Tässä väitöskirjatyössä on selvitetty 1) sisäkorvan epiteelin ja sitä ympäröivän mesenkyymin vuoropuhelun sekä negatiivisten solusyklin säätelijöiden roolia sisäkorvan muotoutumisessa ja 2) negatiivisten solusyklin säätelijöiden roolia nisäkkäiden karvasolujen postmitoottisen tilan ylläpitämisessä. Tässä työssä osoitamme, kuinka sisäkorvan epiteelissä ilmentyvä fibroblastinen kasvutekijä 9 vuoropuhelee ympäröivässä mesenkyymissä sijaitsevien reseptoreidensa kanssa vaikuttaen näin sisäkorvan muotoutumiseen. Sykliinistä riippuva kinaasi-inhiibiittori (CKI) perhe (p21Cip1, p27Kip1 and p57Kip2) sekä retinoblastoma (Rb) perhe (Rb, p107, 130) muodostavat osan negatiivisista solusyklin säätelijöistä. CKI perheen jäsenet ilmentyvät kehittyvän sisäkorvan epiteelissä rajoittaen solunjakaantumista alueittain. Tämä mahdollistaa etenkin tasapainoelinten monimutkaisen muotoutumisen. Tämä työ osoittaa myös, kuinka negatiiviset solusyklin säätelijät ylläpitävät karvasolujen postmitoottista tilaa. Geenien ilmentymisen perusteella perehdyimme retinoblastoma geenin (Rb) sekä geenien p19Ink4d ja p21Cip1 rooliin karvasolujen postmitoottisen tilan säätelyssä. Retinoblastoma geenin inaktivaatio johtaa karvasolujen esiasteiden määrän kasvuun terminaalimitoosien viivästyessä ja normaalisti postmitoottisten karvasolujen jakaantumiseen. Näiden tekijöiden seurauksena sisäkorvassa muodostuu ylimääräisiä karvasoluja. Toisten negatiivisten solusyklin säätelijägeenien, p19Ink4d ja p21Cip1, toiminnan esto johtaa postmitoottisten karvasolujen jakaantumiseen syntymän jälkeisessä kuuloelimessä. Kaikissa edellä mainituissa tapauksissa ylimääräiset karvasolut kuitenkin DNA vaurion seurauksena kuolevat. Nämä tulokset osoittavat kuinka karvasolujen postmitoottista tilaa säätelevät useat eri negatiiviset solusyklin säätelijät. Näiden säätelijöiden normaali toiminta on myös edellytys karvasolujen elinkyvylle. Ymmärrys karvasolujen postmitoottisen tilan ylläpidosta auttaa kehittämään karvasolujen regeneratiivista terapiaa

Cell Cycle Regulation of Stem Cells by MicroRNAs

MicroRNAs (miRNAs) are a class of small non-coding RNA molecules involved in the regulation of gene expression. They are involved in the fine-tuning of fundamental biological processes such as proliferation, differentiation, survival and apoptosis in many cell types. Emerging evidence suggests that miRNAs regulate critical pathways involved in stem cell function. Several miRNAs have been suggested to target transcripts that directly or indirectly coordinate the cell cycle progression of stem cells. Moreover, previous studies have shown that altered expression levels of miRNAs can contribute to pathological conditions, such as cancer, due to the loss of cell cycle regulation. However, the precise mechanism underlying miRNA-mediated regulation of cell cycle in stem cells is still incompletely understood. In this review, we discuss current knowledge of miRNAs regulatory role in cell cycle progression of stem cells. We describe how specific miRNAs may control cell cycle associated molecules and checkpoints in embryonic, somatic and cancer stem cells. We further outline how these miRNAs could be regulated to influence cell cycle progression in stem cells as a potential clinical application