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Research Fellow

Alexander Kuzin, Ph.D.


Room 1B1016
35 CONVENT DR
Bethesda MD 20892
Office: 301-496-8410

Fax: 301-496-1339
kuzina@mail.nih.gov



Our studies have shown that enhancers are made up of multiple conserved sequence blocks (CSBs). Mutagenesis of the nerfin-1 neuroblast enhancer reveals that although each of its CSBs is required for wild-type expression, neither position nor orientation of the CSBs within the enhancer is crucial for enhancer function. While all three conserved E-box transcription factor (TF) binding sites (CAGCTG) are required for full function, adding an additional site interferes with enhancer activity. Our work points to an unexpectedly large number of TFs required for optimal enhancer function - mutant TF analysis has identified at least four that are required for full enhancer regulation.

I have also characterized enhancers regulating the temporal neuroblast determinantcastor, identifying seven enhancers that independently activate reporter/transgene expression in specific sub-patterns of the wild-type gene expression domain. Comparative analysis of the conserved DNA reveals that cas enhancers share different combinations of sequence elements and many of these shared elements contain core DNA-binding recognition motifs for characterized temporal-identity TFs. These studies represent a starting point for the further analysis of cas gene expression and the TFs that regulate it.

Phylogenetic footprinting via EvoPrinter has revealed that cis-regulatory enhancers consist of conserved DNA sequence clusters (CSCs). We report the development of a systematic approach for enhancer discovery. We have generated a Drosophila genome-wide database of conserved DNA consisting of greater than 77,000 CSCs derived from EvoPrintsspanning over 90% of the genome. cis-Decoder database search and alignment algorithms enable the discovery of functionally related enhancers. To demonstrate the utility of these tools, one of the cas neuroblast enhancers was used as input to search for other structurally and functionally related enhancers. The database and accompanying algorithms should prove useful in the discovery and analysis of enhancers involved in any developmental process.



nerfin-1 axon guidance phenotype

Kuzin_Research

Loss-of-function nerfin-1 mutations were generated by the 'ends-in' homologous recombination gene knockout technique. The targeted gene knockout resulted in the isolation of five independent embryonic recessive lethal alleles. Although late-stage homozygous mutant embryos appeared normal, with no detectable gross morphological or segmentation defects, they failed to hatch from their egg chambers. To confirm that the lethality and cellular phenotype observed in the mutant embryos was due to the loss of nerfin-1, independent P-element insertions that contained an 11,154 bp nerfin-1 genomic DNA fragment were used to rescue the viability and cellular phenotype and to restore the nerfin-1 expression level.

Given the absence of any detectable alteration in NB-lineage development in nerfin-1 null embryos, we sought to determine if Nerfin-1 exhibited axon guidance phenotypes. Immunostains of nerfin-1 null embryos revealed significant alterations in axon projections within the embryonic CNS but not in the PNS. For example, within the ventral nerve cord of stage 13 and older nerfin-1 null embryos, the longitudinal connective axon fascicles were disrupted between segments, and both the anterior and posterior commissures of each ventral cord ganglia were malformed (see accompanying figure). Axons that normally project through fascicles that make up the intersegmental longitudinal connectives appeared to either stall or randomly turn at or near segmental boundaries, creating disorganized tangles. In addition, the organization of longitudinal connectives within each of the segments was abnormal with misrouted axons projecting laterally away from the longitudinal tracks. In stage 15 and older mutants, the medial, intermediate, and lateral longitudinal tracks were disrupted along the entire length of the ventral cord.

In contrast to the axon fascicle organization defects observed in the ventral cord and brain, no significant patterning defects were detected in the motoneuron nerve tracts that exit the CNS. In addition, the axon patterning of PNS neurons, outside the CNS, also appeared normal in nerfin-1 null embryos.

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  • 1) Kuzin A, Kundu M, Ross J, Koizumi K, Brody T, Odenwald WF. (2012)
  • The cis-regulatory dynamics of the Drosophila CNS determinant castor are controlled by multiple sub-pattern enhancers
  • Gene Expr Patterns, 12(7-8), 261-72
  • 2) Brody T, Yavatkar AS, Kuzin A, Kundu M, Tyson LJ, Ross J, Lin TY, Lee CH, Awasaki T, Lee T, Odenwald WF. (2012)
  • Use of a Drosophila genome-wide conserved sequence database to identify functionally related cis-regulatory enhancers
  • Dev Dyn. , 241(1), 169-89
  • 3) Kuzin A, Kundu M, Brody T, Odenwald WF (2011)
  • Functional analysis of conserved sequences within a temporally restricted neural precursor cell enhancer.
  • Neural Cell-Fate Determinants Section
  • 4) Kuzin A, Kundu M, Ekatomatis A, Brody T, Odenwald WF. (2009)
  • Conserved sequence block clustering and flanking inter-cluster flexibility delineate enhancers that regulate nerfin-1 expression during Drosophila CNS development
  • Gene Expr Patterns , 9, 65-72
  • 5) Brody T, Yavatkar AS, Lin Y, Ross J, Kuzin A, Kundu M, Fann Y, Odenwald WF. (2008)
  • Horizontal gene transfers link a human MRSA pathogen to contagious bovine mastitis bacteria.
  • PLoS One, 3, e3074
  • 6) Brody T, Rasband W, Baler K, Kuzin A, Kundu M, Odenwald WF (2008)
  • Sequence conservation and combinatorial complexity of Drosophila neural precursor cell enhancers.
  • BMC Genomics, 9, 371
  • 7) Kuzin A, Kundu M, Brody T, Odenwald WF. (2007)
  • The Drosophila nerfin-1 mRNA requires multiple microRNAs to regulate its spatial and temporal translation dynamics in the developing nervous system.
  • Dev Biol, 310, 35-43
  • 8) Odenwald WF, Rasband W, Kuzin A, Brody T (2005)
  • EVOPRINTER, a multigenomic comparative tool for rapid identification of functionally important DNA
  • Proc Natl Acad Sci, 102, 14700-5
  • 9) Kuzin A, Brody T, Moore AW, Odenwald WF. (2005)
  • Nerfin-1 is required for early axon guidance decisions in the developing Drosophila CNS
  • Dev Biol, 277(2), 347-65
  • 10) Kuzin A, Brody T, Moore AW, Odenwald WF (2005)
  • Nerfin-1 is required for early axon guidance decisions in the developing Drosophila CNS
  • Dev Biol, 277, 347-65
  • 11) Broihier HT, Kuzin A, Zhu Y, Odenwald W, Skeath JB (2004)
  • Drosophila homeodomain protein Nkx6 coordinates motoneuron subtype identity and axonogenesis
  • Development, 131, 5233-42
  • 12) Broihier HT, Kuzin A, Zhu Y, Odenwald W, Skeath JB (2004)
  • Drosophila homeodomain protein Nkx6 coordinates motoneuron subtype identity and axonogenesis
  • Development, 131, 5233-42
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