13 research outputs found
Molecular Mechanisms of Androgen Receptor Function In Vivo
Androgens are steroid hormones that regulate the development and function of male reproductive organs as well as physiology of many non-reproductive tissues, such as muscle, bone, liver, and kidney. Moreover, androgen signaling is involved in several pathological conditions, most common of which is prostate cancer.
In its target cells, testosterone or its more potent metabolite 5α-dihydrotestosterone regulates cellular processes by modulating gene expression through the androgen receptor (AR). Ligand-activated AR translocates to nucleus and binds to specific DNA sequences, called androgen response elements (AREs), at the regulatory regions of its target genes. AR cistromes, i.e., global maps of genomic AR occupancy, comprise thousands of AR-binding events primarily located at distal enhancers. AR-binding sites are characterized by distinct histone modifications, and AR recruitment is primed by pioneer factors capable of binding to compact chromatin. AR interacts with a plethora of coregulatory proteins that modify the local chromatin environment and interact with basal transcription machinery. These elements create the complex cellular landscape for androgen action.
The purpose of this thesis was to study molecular determinants of context-specific AR functions in vivo in murine androgen-responsive tissues. The advantage of in vivo studies is that unlike in cancer cell models androgen target cells reside within their physiological environment with an intact AR pathway. In the first part of this work, an androgen reporter mouse line with the luciferase gene under androgenic control was created. In this model, luciferase activity is a measure of AR function, and it can be used for assessing in vivo effects of chemical compounds on AR signaling. The androgen reporter mice were treated with genistein, a phytoestrogenic compound to which people consuming soy products are also exposed. Previous studies have implied that genistein plays a potential role in prostate cancer prevention. The results in this thesis work showed that genistein exhibits tissue-specific effects on AR signaling in vivo. Furthermore, genistein modulates endogenous AR-mediated gene expression in prostate, supporting its potentially beneficial role in prostate carcinogenesis.
In the second part of this work, genomic AR occupancy was examined using chromatin immunoprecipitation (ChIP) coupled with massively parallel sequencing (ChIP-seq). Distinct AR cistromes were identified in three androgen-responsive tissues: prostate, kidney, and epididymis. AR-binding events associate with tissue-specific transcription programs responsible for distinct physiological functions of androgens in these tissues. The key finding in this work was that tissue-specific AR binding is directed by divergent pioneer factors, and that previously identified forkhead box protein A1 (FoxA1) is prostate-specific rather than general pioneer factor for AR. Two novel pioneer factors for AR were identified in this study hepatocyte nuclear factor 4 alpha (Hnf4α) in murine kidney and activating protein 2 alpha (AP-2α) in murine epididymis.
ChIP-seq was also utilized to study in vivo role and characteristics of selective AREs cis-elements not bound by other steroid receptors. Transgenic SPARKI mice have the second zinc finger of the AR DNA-binding domain swapped with the respective part of glucocorticoid receptor, resulting in a chimeric AR unable to bind to selective AREs. A significant proportion of in vivo binding events of wild-type AR were not shared by SPARKI AR in prostate and epididymis, highlighting the importance of selective AREs in AR-specific functions in vivo. Differential receptor binding was also linked to differentially expressed genes in the epididymides of wild-type and SPARKI mice. De novo sequence analysis revealed that the selective AREs are characterized by decreased sequence conversation, indicating that, counter-intuitively, AR selectivity in vivo is achieved by relaxed rather than increased cis-element stringency.
In conclusion, both the AREs and the collaborating proteins contribute to precise AR-regulated transcriptional outcome in the context of native chromatin and distinct tissues. Overall, the results clarified several molecular mechanisms employed by AR in vivo that can potentially contribute to the development of better treatments and diagnostic tools for hormone-dependent disorders in the future.Steroidihormoneihin kuuluvat androgeenit säätelevät sekä miehen lisääntymiselinten kehittymistä ja toimintaa että useiden muiden kudosten, kuten munuaisten, lihasten ja luiden fysiologiaa. Normaalien fysiologisten säätelytehtäviensä lisäksi androgeenit vaikuttavat myös useiden sairauksien kehittymiseen, joista yleisin on eturauhassyöpä.
Androgeenit muokkaavat kohdesolujensa toimintaa säätelemällä geenien luentaa androgeenireseptorin (AR) välityksellä. AR sitoutuu kohdegeeniensä säätelyalueille tunnistaen tietyn DNA-jakson, jota kutsutaan androgeenien vaste-elementiksi. AR:n vaikutus geenien luentaan välittyy vuorovaikutuksessa muiden säätelyproteiinien kanssa, ja yhdessä nämä muuttavat kromatiinin rakennetta ja toimivat transkriptiokoneiston kanssa. Vaikka androgeenien fysiologiset vaikutukset eri kudoksissa tunnetaan melko hyvin, kudosspesifisen hormonivasteen molekyylimekanismit eivät ole vielä selviä.
Tässä väitöskirjassa AR:n toimintaa tutkittiin hiiren androgeenivasteisissa kudoksissa erilaisten hiirimallien ja modernien menetelmien avulla. Kromatiini-immunosaostukseen yhdistetyllä massiivisen rinnakkaisella DNA:n sekvensoinnilla (ChIP-seq) voidaan analysoida transkriptiotekijän DNA:n sitomispaikkoja koko genomin laajuudessa. Syöpäsoluviljelmiin verrattuna hiirikokeiden vahvuus on niiden mahdollistama AR:n vaikutusten tutkiminen fysiologisessa, monia solutyyppejä sisältävässä toimintaympäristössä.
Tässä väitöskirjatyössä valmistettiin uusi siirtogeeninen hiirimalli, jossa androgeenit säätelevät ns. raportoijageenin ilmentymistä. Raportoijageenin aktiivisuutta mittaamalla voidaan tutkia erilaisten kemiallisten yhdisteiden vaikutusta AR:n toimintaan elävän hiiren kudoksissa. Raportoijahiiret altistettiin kasviestrogeeni genisteiinille, jota myös ihmiset saavat soijavalmisteita sisältävästä ravinnosta. Aiempien tutkimustulosten mukaan genisteiini saattaa alentaa riskiä sairastua eturauhassyöpään. Tämän työn tulokset osoittivat, että genisteiini vaikuttaa AR:n signalointiin kudosspesifisellä tavalla. Lisäksi havaittiin genisteiinin säätelevän AR-riippuvaisten geenien aktiivisuutta eturauhasessa, mikä tukee hypoteesia sen vaikutuksista eturauhassyövän kehittymiseen.
Androgeenialtistuksen aikaansaamat AR:n genominlaajuiset sitoutumisprofiilit havaittiin hyvin erilaisiksi hiiren eturauhasessa, munuaisessa ja lisäkiveksessä. AR:n sitoutumispaikat liittyivät kudoskohtaisten geenien ilmentymiseen, selittäen androgeenien erilaisia fysiologisia vaikutuksia näissä kudoksissa. Aiemmissa soluviljelytutkimuksissa on havaittu FoxA1-proteiinin toimivan AR:n pioneeritekijänä, eli sen ajatellaan avaavan tiukkaan paketoitua kromatiinirakennetta AR:n sitoutumista varten. Yksi tämän työn tärkeistä havainnoista on, että AR:n sitoutumista ohjaavat eri pioneeritekijät eri kudoksissa ja että FoxA1:n vaikutus rajoittuu eturauhaseen. Tässä työssä tunnistettiin kaksi uutta pioneeritekijää AR:lle: Hnf4α munuaisessa ja AP-2α lisäkiveksessä.
Muut steroidireseptorit eivät sitoudu AR-selektiivisiin vaste-elementteihin in vitro-olosuhteissa, ja tässä työssä niitä tutkittiin in vivo siirtogeenisen SPARKI-hiiren ja ChIP-seq -menetelmän avulla. SPARKI-hiiren AR on kimeerinen proteiini, jonka DNA:ta sitovan osan toinen sinkkisormi on vaihdettu glukokortikoidireseptorin vastaavaan osaan, minkä johdosta SPARKI-AR ei sitoudu AR-selektiivisiin elementteihin. Merkittävä osa villityypin AR:n genominlaajuisista sitoutumispaikoista eturauhasessa ja lisäkiveksessä todettiin AR-selektiivisiksi in vivo -olosuhteissa. Yksi tämän työn mielenkiintoisista havainnoista on, että AR-selektiivisen DNA-sitoutumisen mahdollistaa reseptorin kyky sitoutua emäsjärjestykseltään vaihtelevampiin vaste-elementteihin kuin muut steroidireseptorit.
Yhteenvetona tässä väitöskirjatyössä osoitettiin, että androgeenien aktivoiman AR:n vuorovaikutus säätelyalueiden vaste-elementtien ja kudosspesifisten pioneeritekijöiden kanssa on edellytys fysiologisesti tarkoituksenmukaiselle geenien ilmentymiselle. Uudet havainnot AR:n toiminnan molekyylimekanismeista voivat tulevaisuudessa mahdollistaa entistä parempien hoitomuotojen kehittämisen ihmisen hormoniriippuvaisiin sairauksiin
A competitive precision CRISPR method to identify the fitness effects of transcription factor binding sites
Here we describe a competitive genome editing method that measures the effect of mutations on molecular functions, based on precision CRISPR editing using template libraries with either the original or altered sequence, and a sequence tag, enabling direct comparison between original and mutated cells. Using the example of the MYC oncogene, we identify important transcriptional targets and show that E-box mutations at MYC target gene promoters reduce cellular fitness. A competitive CRISPR method discovers targets and phenotypic effects of the MYC oncogene.Peer reviewe
A competitive precision CRISPR method to identify the fitness effects of transcription factor binding sites
Here we describe a competitive genome editing method that measures the effect of mutations on molecular functions, based on precision CRISPR editing using template libraries with either the original or altered sequence, and a sequence tag, enabling direct comparison between original and mutated cells. Using the example of the MYC oncogene, we identify important transcriptional targets and show that E-box mutations at MYC target gene promoters reduce cellular fitness. A competitive CRISPR method discovers targets and phenotypic effects of the MYC oncogene.</p
Identification of several potential chromatin binding sites of HOXB7 and its downstream target genes in breast cancer
Peer reviewe
Sequence determinants of human gene regulatory elements
Analysis of massively parallel reporter assays measuring the transcriptional activity of DNA sequences indicates that most transcription factor (TF) activity is additive and does not rely on specific TF-TF interactions. Individual TFs can have different gene regulatory activities. DNA can determine where and when genes are expressed, but the full set of sequence determinants that control gene expression is unknown. Here, we measured the transcriptional activity of DNA sequences that represent an similar to 100 times larger sequence space than the human genome using massively parallel reporter assays (MPRAs). Machine learning models revealed that transcription factors (TFs) generally act in an additive manner with weak grammar and that most enhancers increase expression from a promoter by a mechanism that does not appear to involve specific TF-TF interactions. The enhancers themselves can be classified into three types: classical, closed chromatin and chromatin dependent. We also show that few TFs are strongly active in a cell, with most activities being similar between cell types. Individual TFs can have multiple gene regulatory activities, including chromatin opening and enhancing, promoting and determining transcription start site (TSS) activity, consistent with the view that the TF binding motif is the key atomic unit of gene expression.Peer reviewe
Characterization of an androgen-responsive, ornithine decarboxylase-related protein in mouse kidney
WNT2 activation through proximal germline deletion predisposes to small intestinal neuroendocrine tumors and intestinal adenocarcinomas
Many hereditary cancer syndromes are associated with an increased risk of small and large intestinal adenocarcinomas. However, conditions bearing a high risk to both adenocarcinomas and neuroendocrine tumors are yet to be described.We studied a family with 16 individuals in four generations affected by a wide spectrum of intestinal tumors, including hyperplastic polyps, adenomas, small intestinal neuroendocrine tumors, and colorectal and small intestinal adenocarcinomas.To assess the genetic susceptibility and understand the novel phenotype, we utilized multiple molecular methods, including whole genome sequencing, RNA sequencing, single cell sequencing, RNA in situ hybridization and organoid culture.We detected a heterozygous deletion at the cystic fibrosis locus (7q31.2) perfectly segregating with the intestinal tumor predisposition in the family. The deletion removes a topologically associating domain border between CFTR and WNT2, aberrantly activating WNT2 in the intestinal epithelium. These consequences suggest that the deletion predisposes to small intestinal neuroendocrine tumors and small and large intestinal adenocarcinomas, and reveals the broad tumorigenic effects of aberrant WNT activation in the human intestine.Peer reviewe
Human cell transformation by combined lineage conversion and oncogene expression
Cancer is the most complex genetic disease known, with mutations implicated in more than 250 genes. However, it is still elusive which specific mutations found in human patients lead to tumorigenesis. Here we show that a combination of oncogenes that is characteristic of liver cancer (CTNNB1, TERT, MYC) induces senescence in human fibroblasts and primary hepatocytes. However, reprogramming fibroblasts to a liver progenitor fate, induced hepatocytes (iHeps), makes them sensitive to transformation by the same oncogenes. The transformed iHeps are highly proliferative, tumorigenic in nude mice, and bear gene expression signatures of liver cancer. These results show that tumorigenesis is triggered by a combination of three elements: the set of driver mutations, the cellular lineage, and the state of differentiation of the cells along the lineage. Our results provide direct support for the role of cell identity as a key determinant in transformation and establish a paradigm for studying the dynamic role of oncogenic drivers in human tumorigenesis.Peer reviewe