Virus-transformed pre-B cells show ordered activation but not inactivation of immunoglobulin gene rearrangement and transcription

Virus-transformed pre-B cells undergo ordered immunoglobulin (Ig) gene rearrangements during culture. We devised a series of highly sensitive polymerase chain reaction assays for Ig gene rearrangement and unrearranged Ig gene segment transcription to study both the possible relationship between these processes in cultured pre-B cells and the role played by heavy (H) chain (mu) protein in regulating gene rearrangement. Our analysis of pre-B cell cultures representing various stages of maturity revealed that transcription of each germline Ig locus precedes or is coincident with its rearrangement. Cell lines containing one functional rearranged H chain allele, however, continue to transcribe and to rearrange the allelic, unrearranged H chain locus. These cell lines appear to initiate but not terminate rearrangement events and therefore provide information about the requirements for activating rearrangement but not about allelic exclusion mechanisms

The Proposed New Kosovo Power Plant: An Unnecessary Burden at an Unresaonable Price

The World Bank is deciding whether to fund construction of a new coal-fired power plant outside Kosovo's capital, despite a 2013 pledge to avoid funding coal projects. Kosovars currently get a majority of their electricity from low-grade lignite coal reserves, but air pollution takes a toll on their health and already causes over 800 premature deaths each year.A report by Institute for Energy Economics and Financial Analysis (IEEFA) and Kosovo Civil Society Consortium for Sustainable Development (KOSID), sheds further light on why another coal-fired power plant in Kosovo would be the wrong direction for the country's development.According to the report "the people of Kosovo will have to drastically reallocate their household budgets to pay for the New Kosovo Power Plant if it is built." For instance, if a number of assumptions, like construction costs and economic growth rates, are not met, it is likely that household electricity costs will rise in the country to over twice the European average. The report also projects that the huge debt burden on banks in a country the size of Kosovo will crowd out other new investment across the country.At a November 2015 TEDx presentation, Visar Azemi, a coordinator at KOSID, described the challenge posed by the coal plant as an opportunity for civil society members, think tanks, media organizations and grassroots activists to come together and collaborate on sustainable future for Kosovo, in line with global developments.The report notes that Kosovo has appealing alternatives, including implementing energy-efficiency measures and installing renewable energy sources, that would be less expensive and stimulate a broader range of economic development

Helix-loop-helix transcription factor E47 activates germ-line immunoglobulin heavy-chain gene transcription and rearrangement in a pre-T-cell line

E47 is a helix-loop-helix transcription factor that binds to sites in the immunoglobulin heavy-chain and κ light-chain gene enhancers. Other proteins of this type are involved in cell-type determination. A possible role for E47 in B-cell development was tested by overexpressing a cDNA encoding E47 in the pre-T-cell line 2017. We found a dramatic activation of a germ-line heavy-chain gene transcript in these stable transfectants and an equally large induction of immunoglobulin D-to-J rearrangement, the first recognized step in B-cell development. Germ-line κ light-chain gene transcription and rearrangement were unaffected, but transcription of the recombination-activating genes RAG-1 and RAG-2 and the lymphoid-specific transcription factor Oct-2 was increased. These T cells did not transcribe their rearranged DJ alleles, however, and failed to progress to the next stage of heavy-chain gene assembly, V-to-DJ rearrangement. Because transcription factor E47 can induce pre-T cells to carry out events of B-cell differentiation, it may be a crucial determinant of the earliest stages of B-cell development

Deletion of Germline Promoter PDβ1 from the TCRβ Locus Causes Hypermethylation that Impairs Dβ1 Recombination by Multiple Mechanisms

AbstractThe role of the germline transcriptional promoter, PDβ1, in V(D)J recombination at the T cell receptor β locus was investigated. Deletion of PDβ1 caused reduced germline transcription and DNA hypermethylation in the Dβ1-Jβ1 region and decreased Dβ1 rearrangement. Analyses of methylation levels surrounding recombination signal sequences (RSS) before, during, and after recombination revealed that under physiological conditions cleavage of hypomethylated alleles was preferred over hypermethylated alleles. Methylation of a specific CpG site within the heptamer of the 3′ Dβ1 RSS was incompatible with cleavage by the V(D)J recombinase. These findings suggest that methylation can regulate V(D)J recombination both at a general level by influencing regional chromatin accessibility and specifically by blocking RSS recognition or cleavage by the V(D)J recombinase

Nucleosomes as carriers of epigenetic memory

A core assumption in chromatin biology is that nucleosomes store and transmit epigenetic memory of gene regulation through cell division. A requirement of this model is that nucleosomes – modified in place in one generation – continue to occupy the same DNA locus in the subsequent generation. In my graduate work, I asked whether nucleosomes remember their position through cell division. To execute my experiments, I developed a synthetic chromatin modifying enzyme that deposited high affinity covalent chromatin modification at a defined locus. The chromatin-modifying enzyme could be delocalized within minutes by the addition of a small molecule antibiotic, and the synthetic chromatin modification could be tracked for several hours after preventing new label deposition. Using this synthetic enzyme, I labeled nucleosomes at the GAL10 locus in S cerevisiae and tracked their position through DNA replication. I found that nucleosomes remained localized through DNA replication, suggesting that nucleosomes can transmit epigenetic. Furthermore, I found that in the absence of the fork associated nucleosome chaperones Mcm2 and Dpb3, nucleosomes did not remain localized, suggesting that mcm2 dpb3 mutants cannot transmit epigenetic memory through chromatin. In addition to exploring the fate of nucleosomes during DNA replication, I tracked nucleosomes through transcription and discovered that contrary to published models, nucleosomes are not translated linearly along an open reading frame during transcription. In the thesis that follows, I discuss the context for our collective intuition about the role of chromatin in transmitting epigenetic memory, and I identify the points at which our collective understanding rests on thin or contradictory data. I then discuss my attempts to develop and apply technology to reach a satisfactory resolution of one of the central unanswered questions in chromatin biology: do nucleosomes remember their position? Lastly I discuss side projects adjacent to chromatin biology that occupied my attention during the slower moments of my core thesis research