Summer-Winter, 2006
Epigenetic mechanisms regulating stem cell differentiation in placenta; study of HIF interactions and histone modifications
Supervisor: Susan Fisher & Emin Maltepe, Department of Cell and Tissue Biology,
Human Development & Embryonic Stem Cell Center
University of California, San Francisco, CA, USA
Historically, histones had been viewed as mere chromatin structural components. Recently, however, it has come to be appreciated that the N-terminal tails of histone proteins can incur various post-translational modifications with profound effects on localized chromatin structure and hence, gene expression. Of these modifications, histone acetylation and methylation are two of the most intensely studied. Of the many residues are available for modification, lysine 9 of histone H3 is one of the best characterized. Its acetylation status, like that of others, is determined by the balance of histone acetyltransferase and histone deacetylase activities. In addition, it can be methylated by the Suv39h1,-2 and G9a methyltransferases. While acetylation generally results in the addition of a single acetyl group, methylation status can range from singly to tri-meyhylated. For example, G9a is responsible for di-methylation, whereas Suv39h1 and -2 are responsible for tri-methylation. We previously demonstrated that HDAC activity decreased in the histone H3-K9 acetylation/ methylation during hypoxia. Preliminary data indicate that HDAC activity and histone di-methylation are compromised, whereas histone tri-methylation is exaggerated under hypoxic conditions in the HIF-1alpha-null MEFs. We propose to study HIF-dependent epigenetic responses during hypoxia and differentiation. We have different cells lines to carry out experiments to look at changes during differentiation and hypoxia treatment such as Hif-1-alpha-null MEFs, Arnt-null TS cells, and cell lines deficient G9a, Suv39h1.
We previously reported that Arnt-null TS cells exhibit reduced HDAC activity and altered HDAC subcellular localization during differentiation. To determine whether Hif-1-alpha-null MEFs exhibit defects in hypoxia-induced epigenetic responses, nuclear extracts from cells exposed to normoxia (N, 21%O2) or hypoxia (H, 2%O2) for 16 hours were isolated by hypotonic lysis followed by high salt extraction of nuclear proteins. HDAC1 and HDAC7 are induced by hypoxic culture in wild type cells, whereas Hif-1-alpha-null MEFs exhibit elevated levels of these enzymes that are not further induced under hypoxic conditions. These results were similar to those observed in differentiating Arnt-null TS cells where increased HDAC protein levels and their aberrant subcellular distribution were due to globally disrupted HDAC activities. To determine whether these changes are associated with impaired epigenetic responses on a global level, histone H3-K9 modification were assessed by immunoblot. The hypoxia leads to decrased H3-K9 acetylation in wild-type MEFs, whereas Hif-1-alpha-null MEFs exhibit constitutively elevated levels of Ac-H3-K9 that were not further modified with hypoxic culture. Also, tri-methylated H3-K9 levels were induced by hypoxia in the wild type, but not mutant cells, which exhibited constitutively elevated levels of this modification. These results indicate that HIF-dependent hypoxia responses are not limited to histone acetylation, but encompass histone methylation as well. To further understand the nature of HIF-dependent epigenetic responses, it is thus critical to define the composition of HIF-interacting chromatin complexes as a function of oxygen tension as well as differentiation.
UCSF Fisher Lab, Şu anki çalışmalar...
Şu anda laboratuarımızda, iki konu üzerinde çalışmalar devam ediyor. Birinci çalışmada insan “trophoplast” plasental hücrelerinin döl yatağını işgal işlergesi (mechanism) araştırılıyor. Cenin gelişimi ceninin (embryo) ana rahmine hızlı bir şekilde tutunmasına ve annenin kan dolaşımına ulaşmasına bağlıdır. İnsan plasentasinin cenine ait kısmını oluşturan “trophoplast” hücreleri bu sorunu geçici olarak tümör benzeri özellikleri sayesinde çözüyorlar. Biz işte “trophoplast” istilasının yapışkan ve “proteolytic” işlergesini açığa kavuşturmaya çalışıyoruz. Bunun yanında bu cenine ait hücrelerin nasıl annenin bağışıklık sisteminden kurtulabildiğini belirlemeye çalışıyoruz. Aynı zamanda, plasenta gelişimindeki bilgimizi kullanarak hamilelikle alakalı yaygın bir sorunu, preeclampsia, anlamaya çalışıyoruz. Bu hayati tehlikelere neden olabilen rahatsızlıkta “cytotrophoblast” istilası hiç beklenmedik şekilde yüzeysel oluyor.
İkinci araştırmamızda, bakterilerin kullandığı tutunum işlergesini (mechanism) çalışıyoruz. Tutunum, tutunma hastalık (infection) gelişiminde ilk önemli adımdır. Bu ilk etkileşimler daha çok alıcılarin (receptor) karbonsu (carbohydrate) kısımlarıyla sağlanır. Biz birkaç teknik geliştirdik, böylece bize karmaşık “glycoprotein” karışımlarında hızlı bir şekilde bakterisel alıcıların belirlenmesini sağlıyor. Bundan sonra, kütle “spectrometry” kullanarak bakterisel alıcı etkinliği gösteren “oligosaccharide”lerin eksiksiz yapısını belirliyoruz. Son zamanlarda, “salivary mucin”in hem bakterileri hem de akyuvarları (leukocytes) bağladığını gösterdik. İşte bu işlerge “enfeksiyon” ile mücadelede önemli bir olanak sunuyor bize.