Developmental regulation and stage conversion from trophozoites to cysts is key to disease transmission in E. histolytica. However, the molecular framework that regulates this aspect of amebic biology is poorly defined. We have identified the first developmentally regulated transcription factor in E. histolytica, a Myb domain gene. Our findings demonstrate that EhMyb-dr regulates expression of a subset of stage-specific genes in E. histolytica and that it does so partly by interacting with a C-rich motif in the promoters of regulated genes. This work is the first identification of a developmentally regulated transcription factor in E. histolytica.
Myb proteins are important regulators and are found widely in eukaryotes. In animals, a major function of Myb proteins is to promote cell proliferation and progression through the cell cycle, particularly during development (Oh and Reddy, 1999
). In contrast, many Myb domain proteins in plants and protists are important for the formation of differentiated cell types, including root hair and pigment producing cells in plants (Ramsay and Glover, 2005
). A G. lamblia
Myb protein regulates encystation (Huang et al., 2008
; Sun et al., 2002
), and in Dictyostelium
Myb proteins are required at multiple stages in development (Fukuzawa et al., 2006
; Guo et al., 1999
As with their biological roles, the domain structure of Myb proteins is also variable. The founding member of this family, c-Myb, contains three imperfect tandem repeats of the ~50aa Myb domain, called R1, R2 and R3 (Saikumar et al., 1990
). However, proteins that contain two repeats or even one Myb repeat have been found and shown to have DNA binding activity (Jin and Martin, 1999
). EhMyb-dr is a member of a the SHAQKY subfamily of Myb proteins, which generally have only one or two Myb domains and share a conserved motif (SH[A
]) (Rose et al., 1999
). This family is commonly represented in plants, where members help regulate such diverse functions as maintenance of circadian rhythms (Wang et al., 1997
) and seed maturation (Rubio-Somoza et al., 2006
). A SHAQKY protein from Dictyostelium
is developmentally regulated and required for expression of pre-stalk genes, demonstrating that this Myb family can be important for differentiation (Fukuzawa et al., 2006
). Hence, the proposed role for EhMyb-dr in regulating expression of stage-specific genes would be consistent with the functions of SHAQKY family of Myb proteins.
While mammalian Myb proteins that contain three Myb domains share a common consensus binding site (C
AACGG), plant proteins with Myb domains have diverse motifs (Jin and Martin, 1999
; Rose et al., 1999
; Grotewold et al., 1991
), most likely due to divergent sequences in the C-terminal α-helix of the Myb domain (Jin and Martin, 1999
). The binding site of a Dictyostelium
SHAQKY Myb protein has been identified; a C-rich region (CACCCCAC) was found to be required for both binding and full activity, although additional sequences 5' of the C-rich region were also required (Fukuzawa et al., 2006
). We have identified CCCCCC as a putative binding site for EhMyb-dr. The similarity of the DNA motifs bound by Dictyostelium
and E. histolytica
Myb SHAQKY proteins is intriguing and may indicate that this family of Myb proteins has a conserved binding motif.
The developmentally regulated Myb protein is expressed at low levels in the trophozoite stage and should thus be available, immediately upon receiving the encystation stimuli, to regulate expression of stage-specific genes. This regulation may be direct through binding of the EhMyb-dr to the CCCCCC motif in the genes of promoters that have it. However, there is also likely indirect or secondary induction, as a number of genes induced by EhMyb-dr overexpression do not have this binding motif. Additionally, a number of genes were identified which contain the C-rich motif in their promoter regions but were not regulated by EhMyb-dr overexpression. These genes may be regulated by other transcription modulators or need cofactors not present in the conditions tested.
In summary, we have identified the first developmentally regulated transcription factor, which is likely to be involved in regulating Entamoeba histolytica stage conversion. Our approach identified the transcriptional regulatory network associated with EhMyb-dr as well as the CCCCCC DNA-binding region with which the EhMyb-dr protein interacts. As overexpression of EhMyb-dr was able to induce only a subset of cyst-specific genes, other transcriptional regulators, along with chromatin remolding factors such as histone acetylases, are likely to be required for stage conversion to occur. Further work in characterizing potential regulators of encystation will be necessary to fully understand the molecular and regulatory framework associated with Entamoeba development. Elucidation of these mechanisms will be useful in designing novel means of inhibiting disease transmission.