Understanding how the genes critical to endocrine pancreas development, as well as β-cell function and proliferation are regulated is essential for the development of novel strategies for the production of insulin- secreting cells, and for improving the efficacy of available transplantable material.

The recent development of several novel experimental techniques is beginning to make the elucidation of the mechanisms behind the control of β-cell critical genes, on a global basis, a feasible and tractable task in mammalian biology. The utilization of these techniques to understand endocrine pancreas development and function is highly novel and potentially extremely significant in advancing our understanding of these processes and is the focus of our research.

Unravelling the β-cell Cistrome
The cis-regulatory factors that regulate gene expression in the β-cell are largely unknown. In addition, currently, two of the major questions facing biologists are ‘how do transcription factors identify their binding sites amongst the huge number of potential binding sites within the genome?” and “how is transcription factor binding translated into gene expression?”.

To address these questions and begin to decipher the cis-regulatory factors regulating gene expression during β-cell development and function we are using the recently developed ChIP-seq technique. This technique employs the power of “next generation” or flow cell sequencing for the identification of DNA obtained from chromatin immunoprecipitation experiments, allowing the global localization of transcription factor binding sites and of epigenetic modifications. As such, we are currently using ChIP-seq to, genome-wide, determine binding sites for transcription factors critical to β-cell development and function, and for the localization of epigenetic histone modifications that are thought to be important determinants of transcription factor binding. In addition, we are using flow cell sequencing based strategies to interrogate the β-cell transcriptome using a combination of Tag-seq and RNA-seq approaches that allow an unprecedented level of insight into gene expression levels and the abundance of different transcriptional variants.

By assessing the binding locations of transcription factors, and the localization of epigenetic marks, in combination with gene expression data we can not only gain insight into the basic biology of how transcription factors recognize appropriate binding sites and how different factors work to modulate target gene expression, but also gain insight into how β-cell fate is established and how β-cell function is regulated at a transcriptional level.

Functional Analysis of Myt3, a New Player in the Transcriptional Networks controlling β-cell Development, Function and Apoptosis
It is clear that both genome wide approaches and more targeted gene specific approaches are necessary to fully understand the transcriptional networks driving endocrine development and β-cell function. One of the transcription factors identified in our analysis of the β-cell transcriptorne is Myt3 (also called Sf18). Myt3 is abundantly expressed in adult islets but to date its role in β-cell development or function has not been characterized. Our work suggests that Myt3 plays an interesting role in β-cell development and function, and specifically in β-cell survival and insulin expression and secretion.

In specific the aims of this project are to fully characterize the expression of Myt3 in pancreas development, to identify the regulatory regions responsible for Myt3’s high level of specificity to pancreatic islets, and to elucidate the role of Myt3 in the transcription network driving insulin expression and secretion.

To achieve these results we intend to use classic techniques such as Immunohisotchemistry, luciferase reporter assays, RNAi based suppression studies, and the generation of Myt3 knock-out mice, as well as, a combination of computational, gene expression profiling and ChIP-qPCR studies to identify Myt3 target genes and regulators.

This work is expected to provide novel information on the regulation and functional significance of Myt3 in β-cell development and function.

Marcal N, Belanger-Jasmin S, Hoffman B, Helgason CD, Dang J & Stifani S. Antagonistic effects of Grg6 and Groucho/TLE Proteins on Brain Factor-1 transcription repression activity and cortical neuron differentiation. MCB. 25(24): 10916-10929, 2005.

Siddiqui AS, Khattra J, Delaney AD, Zhao Y, Astell C, Asano J, Babakaiff, Barber S, Beland J, Bohacec S, Brown-John M, Chand S, Charters AM, Cullum R, Dhalla N, Featherstone R, Gerhard DS, Hoffman B, Holt R, Hou J, Kuo B Y-L, Lee Lisa LC, Lee S, Leung D, Ma K, Matsuo C, Mayo M, McDonald H, Prabhu A-L, Pandoh P, Riggins GJ, Ruiz de Algara T, Rupert JL, Smailus D, Stott J, Tsai M, Varhol R, Vrljicak P, Wong D, Wu MK, Xie Yuan-Yun, Yang G, Zhang I, Hirst M, Jones SJM, Helgason CD, Simpson EM, Hoodless PA, Marra Marco. A mouse atlas of Gene Expression: A public, large-scale, digital gene expression profiling resource from precisely defined developing C57BL/6J mouse tissues and cells. PNAS. 102(51): 18485-18490, 2005. This paper was listed as one of the "top 20" most viewed papers in PNAS.

Zhang T*, Hoffman BG*, Ruiz de Algara T, & Helgason CD. SAGE reveals expression of Wnt signalling pathway members during mouse prostate development. Gene Expression Patterns (PMID: 16378759); 6(3): 310-324, 2006. *Co-first authors

Hoffman B, Williams K, Tien AH, Lu V, Ruiz de Algara T, Ting JPY & Helgason CD. Identification of Novel Genes and Transcription Factors Involved in Spleen, Thymus, and Immunological Development and Function. Genes and Immunity (PMID: 16355110); 7(2): 101-112, 2006.

Tucker CA, Kapanen AI, Chikh G, Hoffman BG, Kyle AH, Masin D, Gascoyne RD, Bally M & Klasa RJ. Silencing bcl-2 in models of Mantle Cell Lymphoma correlates with a loss in cyclin D1a expression but not cyclin D1b and is associated with decreases in NFkappaB,p53, bax and p27 levels. Molecular Cancer Theraputics; 7: 749-758, 2008.

Hoffman BG, Zavaglia B, Witzsche J, Ruiz de Algara T, Thiessen N, Bilenky M, Hoodless PA, Jones S, Marra M, & Helgason CD. Identification of Transcripts with Enriched Expression in the Developing and Adult Pancreas. Genome Biology; 9(6): R99,June 14, 2008.

Hoffman BG, Zavaglia B, Witzsche J, Beach M, & Helgason CD. Expression of the Groucho/Tle proteins during pancreas development. BMC Dev. Biol; Sep 8, 8:81, 2008. Highly Accessed.

Morozova O, Morozova V, Hoffman BG, Helagason CD, Marra MA, A seriation approach for visualization-driven discovery of co-expression patterns in serial analysis of gene expression (SAGE) data. PLoS ONE; Sep 12;3(9):e3205, 2008.

Hoffman BG, Jones S. Application of ChIP-Seq to the investigation of transcriptional networks in endocrine organs. Journal of Endocrinology. 201. 2009.

Hoffman BG, Robertson G, Zavaglia B, Beach M, Cullum R, Lee S, Soukhatcheva G, Li L, Wederell ED, Thiessen N, Bilenky M, Cezard T, Tam A, Kamoh B, Birol I, Dai D, Zhao Y, Hirst M, Verchere B, Helgason CD, Marra MA, Jones SJM, Hoodless PA. Locus co-occupancy, nucleosome positioning, and H3K4me1 regulate the functionality of FOXA2-, HNF4A-, and PDX1-bound loci in islets and liver. Genome Research; 20(8): 1037-1051. Listed as Recommended by the Faculty of 1000.

Kim PTW, Hoffman BG, Helgason CD, Verchere CB, Chung SW, Warnock GL, Mui ALF, & Ong CJ. Differentiation of Mouse Embryonic Stem Cells into Endoderm without Embryoid Body Formation. PLoS ONE 5(11): e14146.

Yang C, Bragagnini C, Szabat M, Kott K, Helgason C, Hoffman BG, Johnson JD. Paracrine signaling loops in adult pancreatic islets: Netrins modulate beta-cell apoptosis via neogenin and Unc5a. Diabetologia epub ahead of print DOI: 10.1007/s00125-010-2012-5.