Molecular genetics

Our main interest with regard to genome stability is to understand how DNA damage checkpoint protein kinases are regulated at the molecular level, and how this in turn affects their interaction with substrates.  Because core elements of these pathways are highly conserved throughout evolution, we are able to employ brewer’s yeast as a simple model organism to complement our studies in human and mouse cells. CHK2-like kinases, mutations in which can lead to elevated cancer risk in humans, are characterized by N-terminal SQ/TQ cluster and FHA domains that function as phosphorylation-dependent protein-protein interaction modules. The yeast Rad53 kinase is a unique member of this family in that it contains a second C-terminal FHA domain. We have found that these two FHA domains have largely overlapping functions in the regulation of Rad53 activation by DNA damage during most cell cycle phases. However, the N-terminal FHA1 domain has important non-redundant functions in Rad53 activation during metaphase, and also in linking activated Rad53 to downstream effectors to regulate DNA replication during S phase. In addition, by isolating binding partners for the yeast Rad53 FHA1 domain and the human CHK2 FHA domain, we have identified yeast Mdt1 and human ASCIZ as novel related DNA damage response proteins. In collaboration with Ming-Daw Tsai's lab at Academia Sinica (Taiwan) we found that the Dun1 kinase FHA domain interacts with an SQ/TQ cluster domain of Rad53 in an unprecedented di-phospho-threonine-specific manner that regulates Dun1 activation by Rad53.  We are continuing to study the regulation and function of Chk2-like kinases and ASCIZ in response to DNA damaging as well as during normal unperturbed cell cycles.

Selected Publications

Pike BL, Yongkiettrakul S, Tsai M-D & Heierhorst J (2003). Diverse but overlapping functions of the two forkhead-associated (FHA) domains in Rad53 checkpoint kinase activation.ﾠ J. Biol. Chem. 278, 30421-30424.

McNees CJ, Conlan LA, Tenis N & Heierhorst J (2005). ASCIZ regulates lesion-specific RAD51 focus formation and apoptosis after methylating DNA damage. EMBO J. 24, 2447-2457.

Lee H, Yuan C, Hammet A, Mahajan A, Wu MR, Chen ESW, Su MI, Heierhorst J & Tsai MD (2008). Diphosphothreonine-specific interaction between an SQ/TQ cluster and an FHA domain in the Rad53-Dun1 kinase cascade. Mol. Cell 30, 767-778.

Hoch NC, Chen ESW, Buckland R, Wang SC, Fazio A, Hammet A, Pellicioli A, Chabes A, Tsai MD & Heierhorst J (2013).  Molecular basis of the essential S phase function of the Rad53 checkpoint kinase.  Mol. Cell. Biol. 33, 3202-3213.

We originally identified ASCIZ (ATM substrate Chk2-interacting Zn2+-finger protein; also known as ATMIN or ZNF822) as a new DNA base damage response protein required for the survival of methylating or oxidative DNA damage.  However, we recently found that the protein also has crucial DNA damage-independent functions as an essential Zinc-finger transcription factor.  Mice that lack ASCIZ die late during development with a range of organ development defects, including most strikingly, a complete absence of lungs.  At the molecular level, ASCIZ seems to function as a highly specific “designated” transcription factor for the expression of the dynein light chain gene, Dynll1. In turn, the DYNLL1 protein can bind to at least 10 TQT motifs in the ASCIZ transcription activation domain, and DYNLL1 binding thereby downregulates the transcriptional activity of ASCIZ in a concentration-dependent manner. In this way, the dual ability of ASCIZ to activate Dynll1 gene expression and to sense free DYNLL1 protein levels enables a simple dynamic feedback loop to adjust DYNLL1 levels to cellular needs.

Conditional deletion of ASCIZ in hematopoietic stem cells in the bone marrow, or specifically targeted to B cell precursors, leads to severely impaired B cell development and severely reduced mature B cell numbers in the periphery.  Importantly, this defect can be “rescued” by ectopic expression of DYNLL1, demonstrating the functional importance of ASCIZ as a Dynll1-transcription factor. We recently also found that this tight functional interaction between ASCIZ and DYNLL1 is highly conserved throughout evolution from flies to mammals. Ongoing projects are directed at determining the detailed mechanisms by which the ASCIZ-DYNLL1 axis regulates normal development and the development and cancer in mouse models.

Selected Publications

McNees CJ, Conlan LA, Tenis N & Heierhorst J (2005). ASCIZ regulates lesion-specific RAD51 focus formation and apoptosis after methylating DNA damage. EMBO J. 24, 2447-2457.

Jurado S, Smyth I, Van Denderen B, Tenis N, Hammet A, Hewiitt K, Ng JL, McNees CJ, Kozlov SV, Oka H, Kobayashi M, Conlan LA, Cole TC, Yamamoto K, Taniguchi Y, Takeda S, Lavin MF & Heierhorst J (2010).  Dual functions of ASCIZ in the DNA base damage response and pulmonary organogenesis.  PLoS Genetics 6, e1001170.

Jurado S, Conlan LA, Baker EK, Ng JL, Tenis N, Hoch NC, Gleeson K, Smeets M, Izon D & Heierhorst J (2012).  ATM Substrate Chk2-interacting Zn2+-finger Jurado S, Gleeson K, O'Donnell K, Izon DJ, Walkley CR, Strasser A, Tarlinton DM & Heierhorst J (2012).  The Zinc-finger protein ASCIZ regulates B cell development via DYNLL1 and Bim.  J. Exp. Med. 209, 1629-1639.

Zaytseva O, Tenis N, Mitchell N, Kanno SI, Yasui A, Heierhorst J & Quinn LM (2014).  The novel zinc finger protein dASCIZ regulates mitosis in Drosophila via an essential role in dynein light chain expression.  Genetics 196, 443-453.