ABSTRACT Background. β1,3 galactosyltransferase (B3GALT5) is responsible for the synthesis of type 1 chain oligosaccharides, including Lewis antigens as sialyl-Lewis a, the epitope of tumor marker CA19.9 and an E-selectin ligand potentially involved in cancer malignancy. Transcription occurs through multiple promoters. In some epithelia it is driven by a weak promoter, known as the native promoter that is epigenetically modulated and sensitive to nuclear factor NF-Y. In some organs of the gastrointestinal tract (as the colon, stomach, pancreas and related cell lines) another stronger promoter is active and named the LTR promoter after its retroviral origin. It was supposed to be regulated through a set of homeoproteins: hepatocyte nuclear factor HNF1α/β and caudal-related homeobox Cdx1/2. Surprisingly, B3GALT5 is strongly down regulated in colon cancer, the LTR transcript is not relevant in the small intestine, and Cdx1/2 were reported absent from a cell line expressing large amount of such transcript. Aims. To elucidate the mechanisms controlling transcription of B3GALT5 through its retroviral LTR promoter, in order to explain the tissue specificity and down-regulation in colon adenocarcinomas, and to understand the evolutionary stabilization of the transposon in some primates. Methods. To this aim, we determined the expression levels of putative transcription factors by western blot and the amounts of B3GALT5 LTR transcript by competitive RT-PCR in cancer tissues and cell lines. Moreover, we silenced HNF1α or β in different cell lines, through an shRNA approach, expressed them in another by permanent cDNA transfection, and treated cells with the DNA demethylating agent 5’-AZA-2’-deoxycitydine and in all cases, we measure the effects on LTR transcript levels. We also evaluated the behavior of the LTR promoter in vitro, through electrophoresis mobility shift and reporter luciferase assays. Results. We found that Cdx1/2 are not detectable in cells and tissues expressing high amount of B3GALT5 LTR transcript, while HNF1α/β are well detectable, but even in cells and cancers expressing very low or undetectable levels of the transcript, which is absent in all cells lacking HNF1α/β. Among them, the cell line MDA-MB-231, upon transfection with HNF1α or β, became able to express B3GALT5 LTR transcript, but a very low levels, similar to those found in colon cancers. Transient silencing of HNF1α in cells expressing both HNF1α and β, has no effect on LTR transcript, while similar silencing of HNF1β in cells expressing HNF1β only, determines strong reduction of the transcript. Cell lines expressing high levels of B3GALT5 LTR transcript are affected by the demethylating agent 5AZA that determines strong down regulation of the transcript, falling down to the amounts found in colon cancers, while HNF1 levels remain unaffected. In vitro, luciferase placed under the control of LTR promoter is more active in cells or clones expressing high HNF1 and low or no LTR transcript than in those expressing low HNF1 and high transcript. The same promoter, when used as a probe in EMSA, forms specific complex with nuclear protein extracted from all cells expressing HNF1, irrespectively of the levels of B3GALT5 LTR transcript. Conclusion. Our results suggest that HNF1α and HNF1β are necessary but not sufficient to drive expression of LTR promoter, while Cdx1/2 are not involved. HNF1α/β play an interchangeable and not cumulative role and are not immediately responsible for cancer down-regulation, which depends on a distal regulatory element(s) active when methylated. The successful insertion and activation of B3GALT5 LTR promoter during evolution depended not only on its HNF1 binding site, but even on such distal element(s) unknown at present.
RIASSUNTO Introduzione. La β1,3 galattosiltransferasi (B3GALT5) è responsabile della sintesi della catena oligosaccaridica di tipo 1, tra cui gli antigeni Lewis come il sialil-Lewis a, epitope del marcatore tumorale CA19.9 e ligando della E-selectina, potenzialmente coinvolto nella malignità tumorale. La sua trascrizione è regolata da molteplici promotori. In alcuni epiteli essa è sotto il controllo di un promotore debole, chiamato nativo, modulato epigeneticamente e tramite il fattore nucleare NF-Y. In alcuni organi e cellule di origine gastrointestinale è attivo inoltre un altro promotore, più forte e chiamato LTR per la sua origine retrovirale, che secondo la letteratura dovrebbe essere regolato attraverso il fattore nucleare epatocitario HNF1α/β e quello analogo al Caudale di drosofila Cdx1/2. Tuttavia la B3GALT5 è repressa nel cancro del colon, il trascritto LTR non è rilevante nell’intestino tenue, e Cdx1/2 risultano assenti in una linea cellulare che esprime grandi quantità di tale trascritto. Scopi. Scoprire i meccanismi che controllano la trascrizione di B3GALT5 attraverso il suo promotore LTR, con l’obiettivo di spiegare la specificità tissutale e la repressione negli adenocarcinomi del colon, nonché di capire il processo di stabilizzazione evolutiva del trasposone in alcuni primati. Metodi. A questo scopo abbiamo quantificato l’espressione di HNF1α/β e Cdx1/2, tramite Western Blot, e quella del trascritto B3GALT5 LTR, tramite RT-PCR competitiva, in tessuti tumorali e linee cellulari. Inoltre, abbiamo silenziato HNF1α o β in diverse cellule, tramite shRNA, e li abbiamo espressi in un’altra, tramite trasfezione del cDNA. Abbiamo quindi trattato delle cellule con l'agente demetilante il DNA 5'-AZA-2'-desossicitidina, misurandone poi i livelli di trascritto LTR. Abbiamo infine valutato il promotore LTR in vitro, mediante saggi di EMSA e di luciferasi. Risultati. Cdx1/2 risultano immisurabili in cellule e tessuti che pur esprimono alti livelli di trascritto LTR, mentre HNF1α/β sono presenti anche in cellule e tumori che esprimono una quantità bassa o nulla del trascritto. Nelle cellule che non esprimono HNF1α/β però non c’è espressione alcuna del trascritto LTR. Tra queste, le MDA-MB-231, dopo transfezione con HNF1α o β, esprimono il trascritto LTR, ma a bassi livelli, paragonabili a quelli dei tumori del colon. Il silenziamento di HNF1α in una linea cellulare che esprime entrambi i fattori HNF1α e β non ha effetti nell’espressione del trascritto LTR, mentre quello di HNF1β in un’altra linea che esprime solo HNF1β produce forte riduzione del trascritto. Pure il trattamento con 5'-AZA-2'-desossicitidina riduce il trascritto in cellule che ne esprimono alti livelli, portandolo ai livelli dei tumori del colon, e senza effetto sulla quantità di HNF1. Usando i saggi delle luciferasi, abbiamo visto che la luciferasi sotto il controllo del promotore LTR è più attiva in cellule e cloni che esprimono alte quantità di HNF1, anche se non esprimono o esprimono poco trascritto LTR, che in quelle cellule che esprimono poco HNF1 ma magari una grande quantità di trascritto. Usando la sequenza del promotore LTR in saggi di EMSA, abbiamo visto che forma dei complessi specifici con estratti di proteine nucleari di tutte le linee cellulari che esprimono HNF1, indipendentemente dei livelli di espressione del trascritto B3GALT5 LTR. Conclusione. I nostri risultati suggeriscono che HNF1α e -β sono necessari ma non sufficienti a regolare l'espressione del promotore LTR, mentre Cdx1/2 non sono coinvolti. HNF1α/β svolgono un ruolo intercambiabile e non cumulativo, e non sono immediatamente responsabili della regolazione negativa che avviene nel cancro, che invece dipende da elementi regolatori distanti attivi solo se metilati. Il successo della inserzione e l'attivazione del promotore B3GALT5 LTR durante l'evoluzione dipendono quindi non solo dal suo sito di legame a HNF1, ma anche da questi elementi distanti attualmente sconosciuti.
TRANSCRIPTIONAL REGULATION OF THE B3GALT5 GENE / A. Zulueta Morales ; tutore: R. Ghidoni ; co-tutore: M. Trinchera ; direttore del dottorato: M. Clerici. DIPARTIMENTO DI SCIENZE DELLA SALUTE, 2014 Jan 27. 26. ciclo, Anno Accademico 2013. [10.13130/zulueta-morales-aida_phd2014-01-27].
TRANSCRIPTIONAL REGULATION OF THE B3GALT5 GENE
A. ZULUETA MORALES
2014
Abstract
ABSTRACT Background. β1,3 galactosyltransferase (B3GALT5) is responsible for the synthesis of type 1 chain oligosaccharides, including Lewis antigens as sialyl-Lewis a, the epitope of tumor marker CA19.9 and an E-selectin ligand potentially involved in cancer malignancy. Transcription occurs through multiple promoters. In some epithelia it is driven by a weak promoter, known as the native promoter that is epigenetically modulated and sensitive to nuclear factor NF-Y. In some organs of the gastrointestinal tract (as the colon, stomach, pancreas and related cell lines) another stronger promoter is active and named the LTR promoter after its retroviral origin. It was supposed to be regulated through a set of homeoproteins: hepatocyte nuclear factor HNF1α/β and caudal-related homeobox Cdx1/2. Surprisingly, B3GALT5 is strongly down regulated in colon cancer, the LTR transcript is not relevant in the small intestine, and Cdx1/2 were reported absent from a cell line expressing large amount of such transcript. Aims. To elucidate the mechanisms controlling transcription of B3GALT5 through its retroviral LTR promoter, in order to explain the tissue specificity and down-regulation in colon adenocarcinomas, and to understand the evolutionary stabilization of the transposon in some primates. Methods. To this aim, we determined the expression levels of putative transcription factors by western blot and the amounts of B3GALT5 LTR transcript by competitive RT-PCR in cancer tissues and cell lines. Moreover, we silenced HNF1α or β in different cell lines, through an shRNA approach, expressed them in another by permanent cDNA transfection, and treated cells with the DNA demethylating agent 5’-AZA-2’-deoxycitydine and in all cases, we measure the effects on LTR transcript levels. We also evaluated the behavior of the LTR promoter in vitro, through electrophoresis mobility shift and reporter luciferase assays. Results. We found that Cdx1/2 are not detectable in cells and tissues expressing high amount of B3GALT5 LTR transcript, while HNF1α/β are well detectable, but even in cells and cancers expressing very low or undetectable levels of the transcript, which is absent in all cells lacking HNF1α/β. Among them, the cell line MDA-MB-231, upon transfection with HNF1α or β, became able to express B3GALT5 LTR transcript, but a very low levels, similar to those found in colon cancers. Transient silencing of HNF1α in cells expressing both HNF1α and β, has no effect on LTR transcript, while similar silencing of HNF1β in cells expressing HNF1β only, determines strong reduction of the transcript. Cell lines expressing high levels of B3GALT5 LTR transcript are affected by the demethylating agent 5AZA that determines strong down regulation of the transcript, falling down to the amounts found in colon cancers, while HNF1 levels remain unaffected. In vitro, luciferase placed under the control of LTR promoter is more active in cells or clones expressing high HNF1 and low or no LTR transcript than in those expressing low HNF1 and high transcript. The same promoter, when used as a probe in EMSA, forms specific complex with nuclear protein extracted from all cells expressing HNF1, irrespectively of the levels of B3GALT5 LTR transcript. Conclusion. Our results suggest that HNF1α and HNF1β are necessary but not sufficient to drive expression of LTR promoter, while Cdx1/2 are not involved. HNF1α/β play an interchangeable and not cumulative role and are not immediately responsible for cancer down-regulation, which depends on a distal regulatory element(s) active when methylated. The successful insertion and activation of B3GALT5 LTR promoter during evolution depended not only on its HNF1 binding site, but even on such distal element(s) unknown at present.File | Dimensione | Formato | |
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