The Opposite Side Line Of The C. Elegans Cbf¦Â Homolog, Bro-1 Cell Proliferation, Differentiation And Fate Determination, Regulation And Control

The RUNX/CBFβheterodimeric transcription factor plays an important role in regulating cell proliferation and differentiation in a variety of developmental contexts. Aberrant function of Runx and CBFβhas been causally related to the development of various diseases,including acute myeloid leukemia,gastric cancer and cleidocranial dysplasia.The underlying mechanism of the RUNX/CBFβcomplex in regulation of cell proliferation is still poorly defined.In this study,a C.elegans CBFβhomolog,bro-1 was cloned and its function in proliferation,differentiation and specification of seam cells was investigated.Also the physical interactions between RNT-1 and BRO-1 as well as the epistasis of RNT/BRO-1 complex in the cell cycle regulation pathway were explored.1.Isolation of mutant bp133 and identification,mapping and cloning of bro-1 Strain JR667(scm::gfp) was used for mutagenesis and F2 progenies were screened. Mutant bp133 has a decreased seam cell number.Three-factor mapping and rescue experiment showed bp133 was a novel allele of C.elegans CBFβhomolog,bro-1. Sequence data suggested that bro-1(bp133) is a nonsense mutation which changes amino acid Trp to a stop codon at the 91^th amino acid.2.Function of bro-1 in regulating seam cell division and differentiation Lineage analysis showed that bro-1 mutation disrupted the proliferative seam cell division at the L2 stage.Furthermore,bro-1 mutation caused serious ray missing and fusion defects in males.Analysis of double mutants between bro-1(bp133) and several heterochronic mutants showed that the proliferative seam cell division occurred at any stage was defective in bro-1 mutation background.Though bro-1 mutation did not affect the seam cell fusion,the development ofalae was defective in bro-1 mutants.After introduced into some tissue specific transgenic markers(elt-2::gfp,lag-2::gfp), bro-1 mutation does not appear to affect those post-embryonic cell divisions.The development of V5 derived postdeirid structure is defective in bro-1 mutants visualized by postdeirid specific marker dat-1::gfp.Meanwhile,DiI assay showed defects in the differentiation of the daughter of T cell.3.Expression pattern of bro-1bro-1:.gfp reporter suggested that bro-1 expressed in both cytoplasm and nucleus of seam cells from bean stage to young adult.The bro-1(bp133)::gfp displayed weak or no expression of GFP.scm::bro-1 which was restrictedly expressed in the seam cells fully rescued bro-1(bp133).Furthermore,abolishment of elt-5/elt-6 by RNAi severely disrupt the expression of bro-1::gfp.4.RNT-1/BRO-1 complex and DNA-binding activity of RNT-1Pull down results suggested that BRO-1 specifically bound to RNT-1 which was mediated by the RUNT domain and other regions of RNT-1 were required for the binding specificity of the RUNT domain.Gel shift experiments showed that RNT-1 directly bound to DNA through its RUNT domain and BRO-1 can elevate the DNA-binding activity of RNT-1.5.Interactions between RNT-1/BRO-1 complex and UNC-37bp138 has a decreased seam cell number as bro-1.Three-factor mapping and PCR product rescue showed that bp138 was a new allele of unc-37.Sequence data showed A Glycine to Glutamine(G to E) mutation at amino acid 393 in bp138.Pull down assay suggested that RNT-1 specifically interacted with UNC-37,while BRO-1 showed no interaction with UNC-37.Further analysis indicated that the RUNT domain of RNT-1 mediated the binding to UNC-37.The interaction domain in UNC-37 was mapped to its WD repeats.6.RNT-1/BRO-1 complex and cell cycle machineryInactivity of cell cycle regulators lin-35;fzr-1 and cki-1 by RNA interference can suppress the seam cell division defects of bro-1 mutants to some extent.Meanwhile extra seam cell divisions occurred in lin-35;fzr-1 double mutants.7.Functions of RNT-1/BRO-1 complex and unc-37 and sop-1 in specifying the seam cell fateSome seam cell fates in bro-1 unc-37 double mutant transformed into other epidermal cell fates which were absent either in bro-1 or unc-37 single mutant.Furthermore,an unc-37 mutation greatly enhanced the defects in the differentiation of the daughters of V5 in bro-1 mutants.Inactivity of sop-1 by RNAi in bro-1(bp133) mutants also caused seam cell fate transformation.Furthermore,mutation in sop-1 caused a synthetic lethal phenotype when coupled with the bro-1 or rnt-1.In conclusion,this study reports a C.elegans CBFβhomologue,bro-1,and elucidates its activity in seam cell proliferation,differentiation and cell fate determination.Meanwhile this study indicates the conserved RNT-1/BRO-1 complex can promote the G1 to S progression of seam cells by regulating the downstream targets.