HERVs, transposons and human diseases : part I

Part 2 and part 3 of the article can be found through this link: https://www.um.edu.mt/library/oar//handle/123456789/13012 https://www.um.edu.mt/library/oar//handle/123456789/13132It has been found that the human genome is full of relic retroviral DNA sequences called HERVs (Human Endogenous RetroViruses). A HERV is a type of a transposon, the latter being a piece of DNA sequence that can move from one position to another position in the genome, hence its other name of ‘jumping gene’. HERVs and other transposons are held in check from doing havoc in the genome by several mechanisms, one of which is epigenetic in nature (namely DNA methylation and histone modifications). HERVs and other transposons are being implicated to have physiological and pathological functions in the genomes of the cells that host them. Accumulating evidence is showing that they may be associated with certain human diseases, specifically in some autoimmune diseases (e.g. rheumatoid arthritis, psoriasis, systemic lupus erythematosus, insulin-dependent diabetes mellitus), neurological diseases (e.g. schizophrenia, multiple sclerosis, motor neuron disease) and cancer. Understanding how these relic viruses and other jumping genes bring about these human diseases could help in their prevention and treatment.peer-reviewe

HERVs, transposons and human diseases : part II

Part 1 and part 3 of the article can be found through this link : https://www.um.edu.mt/library/oar//handle/123456789/12961 https://www.um.edu.mt/library/oar//handle/123456789/13132Part 2 of the article. It has been found that the human genome is full of relic retroviral DNA sequences called HERVs (Human Endogenous RetroViruses). A HERV is a type of a transposon, the latter being a piece of DNA sequence that can move from one position to another position in the genome, hence its other name of ‘jumping gene’. HERVs and other transposons are held in check from doing havoc in the genome by several mechanisms, one of which is epigenetic in nature (namely DNA methylation and histone modifications). HERVs and other transposons are being implicated to have physiological and pathological functions in the genomes of the cells that host them. Accumulating evidence is showing that they may be associated with certain human diseases, specifically in some autoimmune diseases (e.g. rheumatoid arthritis, psoriasis, systemic lupus erythematosus, insulin-dependent diabetes mellitus), neurological diseases (e.g. schizophrenia, multiple sclerosis, motor neuron disease) and cancer. Understanding how these relic viruses and other jumping genes bring about these human diseases could help in their prevention and treatment.peer-reviewe

HERVs, transposons and human diseases : part 3

Part 1 and 2 of this article can be found through theses links: https://www.um.edu.mt/library/oar//handle/123456789/12961 https://www.um.edu.mt/library/oar//handle/123456789/13012Part 3 of the article. It has been found that the human genome is full of relic retroviral DNA sequences called HERVs (Human Endogenous RetroViruses). A HERV is a type of a transposon, the latter being a piece of DNA sequence that can move from one position to another position in the genome, hence its other name of ‘jumping gene’. HERVs and other transposons are held in check from doing havoc in the genome by several mechanisms, one of which is epigenetic in nature (namely DNA methylation and histone modifications). HERVs and other transposons are being implicated to have physiological and pathological functions in the genomes of the cells that host them. Accumulating evidence is showing that they may be associated with certain human diseases, specifically in some autoimmune diseases (e.g. rheumatoid arthritis, psoriasis, systemic lupus erythematosus, insulin-dependent diabetes mellitus), neurological diseases (e.g. schizophrenia, multiple sclerosis, motor neuron disease) and cancer. Understanding how these relic viruses and other jumping genes bring about these human diseases could help in their prevention and treatment.peer-reviewe

Human endogenous retrovirus K106 (HERV-K106) was infectious after the emergence of anatomically modern humans.

HERV-K113 and HERV-K115 have been considered to be among the youngest HERVs because they are the only known full-length proviruses that are insertionally polymorphic and maintain the open reading frames of their coding genes. However, recent data suggest that HERV-K113 is at least 800,000 years old, and HERV-K115 even older. A systematic study of HERV-K HML2 members to identify HERVs that may have infected the human genome in the more recent evolutionary past is lacking. Therefore, we sought to determine how recently HERVs were exogenous and infectious by examining sequence variation in the long terminal repeat (LTR) regions of all full-length HERV-K loci. We used the traditional method of inter-LTR comparison to analyze all full length HERV-Ks and determined that two insertions, HERV-K106 and HERV-K116 have no differences between their 5' and 3' LTR sequences, suggesting that these insertions were endogenized in the recent evolutionary past. Among these insertions with no sequence differences between their LTR regions, HERV-K106 had the most intact viral sequence structure. Coalescent analysis of HERV-K106 3' LTR sequences representing 51 ethnically diverse individuals suggests that HERV-K106 integrated into the human germ line approximately 150,000 years ago, after the emergence of anatomically modern humans

TRIM28-Regulated Transposon Repression Is Required for Human Germline Competency and Not Primed or Naive Human Pluripotency.

Transition from primed to naive pluripotency is associated with dynamic changes in transposable element (TE) expression and demethylation of imprinting control regions (ICRs). In mouse, ICR methylation and TE expression are each regulated by TRIM28; however, the role of TRIM28 in humans is less clear. Here, we show that a null mutation in TRIM28 causes significant alterations in TE expression in both the naive and primed states of human pluripotency, and phenotypically this has limited effects on self-renewal, instead causing a loss of germline competency. Furthermore, we discovered that TRIM28 regulates paternal ICR methylation and chromatin accessibility in the primed state, with no effects on maternal ICRs. Taken together, our study shows that abnormal TE expression is tolerated by self-renewing human pluripotent cells, whereas germline competency is not

Vaccination directed against the human endogenous retrovirus-K envelope protein inhibits tumor growth in a murine model system

Human endogenous retrovirus (HERV) genomes are chromosomally integrated in all cells of an individual. They are normally transcriptionally silenced and transmitted only vertically. Enhanced expression of HERV-K accompanied by the emergence of anti-HERV-K-directed immune responses has been observed in tumor patients and HIV-infected individuals. As HERV-K is usually not expressed and immunological tolerance development is unlikely, it is an appropriate target for the development of immunotherapies. We generated a recombinant vaccinia virus (MVA-HKenv) expressing the HERV-K envelope glycoprotein (ENV), based on the modified vaccinia virus Ankara (MVA), and established an animal model to test its vaccination efficacy. Murine renal carcinoma cells (Renca) were genetically altered to express E. coli beta-galactosidase (RLZ cells) or the HERV-K ENV gene (RLZ-HKenv cells). Intravenous injection of RLZ-HKenv cells into syngenic BALB/c mice led to the formation of pulmonary metastases, which were detectable by X-gal staining. A single vaccination of tumor-bearing mice with MVA-HKenv drastically reduced the number of pulmonary RLZ-HKenv tumor nodules compared to vaccination with wild-type MVA. Prophylactic vaccination of mice with MVA-HKenv precluded the formation of RLZ-HKenv tumor nodules, whereas wild-type MVA-vaccinated animals succumbed to metastasis. Protection from tumor formation correlated with enhanced HERV-K ENV-specific killing activity of splenocytes. These data demonstrate for the first time that HERV-K ENV is a useful target for vaccine development and might offer new treatment opportunities for diverse types of cancer

Evidence of extensive non-allelic gene conversion among LTR elements in the human genome

Long Terminal Repeats (LTRs) are nearly identical DNA sequences found at either end of Human Endogenous Retroviruses (HERVs). The high sequence similarity that exists among different LTRs suggests they could be substrate of ectopic gene conversion events. To understand the extent to which gene conversion occurs and to gain new insights into the evolutionary history of these elements in humans, we performed an intra-species phylogenetic study of 52 LTRs on different unrelated Y chromosomes. From this analysis, we obtained direct evidence that demonstrates the occurrence of ectopic gene conversion in several LTRs, with donor sequences located on both sex chromosomes and autosomes. We also found that some of these elements are characterized by an extremely high density of polymorphisms, showing one of the highest nucleotide diversities in the human genome, as well as a complex patchwork of sequences derived from different LTRs. Finally, we highlighted the limits of current short-read NGS studies in the analysis of genetic diversity of the LTRs in the human genome. In conclusion, our comparative re-sequencing analysis revealed that ectopic gene conversion is a common event in the evolution of LTR elements, suggesting complex genetic links among LTRs from different chromosomes

Expression and regulation of human endogenous retroviruses (HERVs) in developing and mature T cells

Tese de mestrado, Biologia (Biologia Molecular e Genética), Universidade de Lisboa, Faculdade de Ciências, 2015The Human genome comprises almost 8% long terminal repeat (LTR)-retroelements, in which Human Endogenous Retroviruses (HERVs) are included. More than 30 HERV groups have been identified. They share a similar provirus structure with exogenous retroviruses, despite harboring many inactivating mutations. Interestingly, HERVs have been increasingly associated with cancer, autoimmunity and infectious diseases. Data on HERV expression in T cells is sparse. Here we investigated, for the first time, the expression of several HERV families during human T cell development and differentiation. HERV-K, -W, -P and -R envelope (env) expression were analyzed in purified T cell subsets from the human thymus, peripheral blood or tonsils using real time RT-PCR. In addition, Env protein expression was studied in thymic and tonsillar tissue using immunohistochemistry. We observed a similar pattern of HERV env transcriptional expression during the development and maturation of thymocytes in the thymus. HERV levels tended to be higher in mature thymocytes than in the early developmental stages, supporting their differential regulation during T cell development. Regarding the peripheral blood compartment, HERVs showed similar transcriptional levels within naïve and memory CD4 and CD8 T cells, with small inter-individual variation. Moreover, HERV expression was modulated during T follicular helper (TFH) differentiation in human tonsils. Importantly, we further confirmed Env protein production in lymphoid tissues, as HERV-K Env protein was expressed in the medullary compartment of the thymus, as well as in the T cell zone of the tonsil. Our data regarding HERV env expression profiles during T cell development and maturation prompted us to test potential pathways of HERV regulation. We demonstrated, using real time RT-PCR, that T cell receptor (TCR) stimulation and Phorbol-12-myristate-13- acetate (PMA)-Ionomycin were able to down-regulate HERV-W, -P and -R transcriptional levels in CD4SP thymocytes. Additionally, a positive correlation was found between HERV env expression and the transcriptional levels of the retroviral restriction factor deoxycytidine deaminases apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) throughout T cell development and differentiation, which was not observed for APOBEC3F. Overall, our data support a tight regulation of HERV expression during T cell development and differentiation. This appears to be particularly pertinent in the thymus, with possible implications for the process of T cell selection.O genoma humano é composto por aproximadamente 8% de retroelementos contendo repetições terminais longas (LTRs), nos quais se incluem os Retrovírus Endógenos Humanos (HERVs). Os HERVs são sequências de ADN que se fixaram no genoma humano após uma primeira infeção da linha germinativa por um retrovírus exógeno, sendo subsequentemente transmitidos verticalmente. A sua diversidade depende das interações com o hospedeiro, no entanto somente alguns HERVs foram capazes de persistir, sofrendo mutações e deleções que resultaram na perda da capacidade de replicação. Os HERVs dividem-se em mais de 30 famílias e 3 classes, de acordo com a sua homologia a retrovírus exógenos, sendo denominados pela letra que define o ácido ribonucleico de transferência (tRNA) usado para a transcrição reversa do seu genoma. Apesar de a maioria dos HERVs estar defetivo, vários estudos demonstraram a expressão de RNA e de proteína em vários tecidos humanos e linhas celulares, e em raras situações a produção de partículas virais intactas. Pensa-se que eles persistiram no genoma humano por duas razões: exercerem funções biológicas ou devido à incapacidade de o hospedeiro os eliminar. Curiosamente, apenas a função das proteínas do invólucro (Env) do HERV-W e HERV-FRD é reconhecida. Por outro lado, o estudo dos HERVs num contexto de doença tem aumentado, uma vez que eles têm sido associados com a ocorrência de cancro, autoimunidade e doenças infeciosas. Como não podemos excluir a existência de um HERV infecioso, é importante perceber o que pode regular a sua expressão. Canonicamente, a sua transcrição depende dos seus promotores. No entanto esta pode ser igualmente ativada por fatores de transcrição celulares (i.e., NF-κB) ou proteínas virais. Posto isto, o hospedeiro desenvolveu mecanismos para controlar a sua expressão. O enzima “apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like” 3G (APOBEC3G) e outros membros da família das citidina desaminases foram descritos como fatores de restrição virais em células humanas, atuando sobre a replicação de retrovírus, tais como o HIV e alguns HERVs. Sabe-se igualmente, que a sua transcrição é reprimida por mecanismos de metilação do DNA, modificação das histonas e RNA de interferência, no entanto os estudos em humanos são limitados. A eficiência do sistema imunitário depende da resposta da imunidade inata e adaptativa para eliminar invasões patogénicas. A imunidade inata representa a primeira linha de defesa, sendo constituída pela barreira epitelial, proteínas solúveis e moléculas bioativas. Subsequentemente, a imunidade adaptativa atua pelo reconhecimento específico de antigénios. Este reconhecimento é feito usando receptores antigénicos expressos à superfície de linfócitos B e T. No entanto, para esta resposta se dar corretamente, as células B e T têm de submeter-se a vários processos de seleção e maturação. Progenitores de células T expressando CD34+ migram da medula óssea para o timo, onde irá ocorrer o desenvolvimento e maturação das células T. Os timócitos serão distinguidos de acordo com a expressão dos corecetores CD4 e CD8, em: timócitos “triple negative” CD34+ (TN CD34+, CD4-CD8-CD3-), timócitos CD4 “immature single positive” (CD4ISP, CD4+CD8-CD3-), timócitos “double positive” (DP, CD4+CD8+CD3-/+), e timócitos CD4 ou CD8 “single positive” (SP, CD4+/-CD8-/+CD3+). Durante o processo de maturação e seleção, os timócitos sofrem rearranjo do recetor de células T (TCR) β e α. Interações de baixa afinidade entre o TCRαβ de timócitos DP e péptidos do próprio complexados com antigénios no complexo major de histocompatibilidade (MHC) são selecionados positivamente. Por outro lado, a seleção negativa ocorre se esta afinidade for demasiado alta. Timócitos selecionados positivamente diferenciam-se em células T CD4SP ou CD8SP, que após maturação migram para a periferia. Na periferia, as células T “naïve” circulam através dos órgãos linfóides secundários (p.ex. amígdalas) até sofrerem ativação, processo que envolve o encontro de antigénios apresentados por células dendríticas e o sinal co-estimulator emitido pelas mesmas (CD28). Após ativação, estas células adquirem uma das funções efetoras de células T auxiliar: Th1, Th2, Th17, Th9, T folicular auxiliar ou T reguladora. Em contrapartida, as células T CD8 “naïve” diferenciam-se em citotóxicas (CTLs). Sabe-se que, 5-10% das células T CD4 e CD8 efetoras persistem como células T de memória, caracterizadas por oferecer uma maior e mais eficiente resposta após segunda exposição antigénica. A expressão dos HERVs em células T tem sido pouco estudada. Neste projeto foi investigado, pela primeira vez, a expressão de várias famílias dos HERVs durante o desenvolvimento e diferenciação de células T. A expressão do gene env do HERV-K, -W, -P e -R foram analisadas em subpopulações de células T purificadas do timo humano, sangue periférico e amígdalas, usando Real Time RT-PCR. Paralelamente, foi avaliada a expressão da proteína Env em tecido do timo e amígdalas usando imunohistoquímica (IHQ). Os resultados revelaram um padrão semelhante de expressão génica dos HERVs durante o desenvolvimento e maturação dos timócitos no timo. Os níveis transcricionais apresentaram uma tendência para serem maiores nas fases mais desenvolvidas (CD4SP e CD8SP) do que nas fases iniciais do desenvolvimento (TN CD34+ e CD4ISP), indicando que possam estar a ser distintamente regulados. Estudos de vários grupos podem explicar este padrão. Conrad e colegas identificaram a expressão no timo humano de superantigénios codificados pelo membro HERV-K18, na fase DP com o intuito de induzir a seleção negativa de timócitos CD4 Vβ7. Adicionalmente, sabe-se que o desenvolvimento de células T no timo é altamente regulado por mecanismos epigenéticos; nas fases iniciais do desenvolvimento o DNA encontra-se hipermetilado, ao passo que ao longo da maturação e seleção o DNA torna-se hipometilado. Os HERVs são reprimidos com a metilação do DNA, por isso é possível especularmos que estes mecanismos possam explicar o padrão de expressão dos HERVs no desenvolvimento dos timócitos. Relativamente à periferia, vários estudos analisaram os níveis de HERVs em células mononucleares totais do sangue periférico. Neste projeto analisamos os seus níveis em subpopulações do sangue periférico. Os HERVs revelaram níveis transcricionais similares entre células T CD4 e CD8 “naïve” e de memória, variando pouco entre os indivíduos analisados. Podemos nesse caso especular que, o processo de proliferação homeostático envolvido na manutenção das populações “naïve” e de memória não afeta a expressão dos HERVs. Adicionalmente, a atividade transcricional dos HERVs parece ser modulada nas amígdalas durante a diferenciação de células T foliculares auxiliares. Apesar do estudo dos HERVs em órgãos linfóides secundários ser inexistente, pensamos que esta modulação se deve à ativação e ação de mecanismos regulatórios que reprimem especificamente a transcrição dos diferentes HERVs durante a diferenciação celular. É importante referir que os resultados da IHQ confirmaram a expressão da proteína Env do HERV-K no timo (medula) e na amígdala (zona das células T), e apoiam os dados obtidos na análise da expressão génica. Os dados gerados durante o desenvolvimento das células T no timo, levou-nos a investigar os possíveis mecanismos de regulação da transcrição dos HERVs. Conseguimos demonstrar, usando Real Time RT-PCR, que a estimulação por via de TCR e “PMAIonomycin” foi capaz de regular negativamente a expressão do HERV-W, -P e -R em timócitos CD4SP. Adicionalmente, observamos uma correlação positiva entre os níveis transcricionais dos HERVs e do fator de restrição APOBEC3G ao longo do desenvolvimento e diferenciação de células T, mas não com o enzima APOBEC3F. Efetivamente, parece que os mecanismos que controlam os níveis dos HERVs são específicos da célula ou população celular em que eles se expressam. Em suma, os nossos resultados indicam que os HERVs são estritamente regulados durante o desenvolvimento e diferenciação das células T. Esta regulação aparenta ser mais relevante no timo, com implicações no processo de seleção das células T