Background Histone methylation is thought to be central to the epigenetic mechanisms that maintain and confine cellular identity in multi-cellular organisms. To examine epigenetic roles in cellular homeostasis, we conditionally mutated the histone 3 lysine 4 methyltransferase, Mll2, in embryonic stem (ES) cells, during development and in adult mice using tamoxifen-induced Cre recombination. Results In ES cells, expression profiling unexpectedly revealed that only one gene, Magoh2, is dependent upon Mll2 and few other genes were affected. Loss of Mll2 caused loss of H3K4me3 at the Magoh2 promoter and concomitant gain of H3K27me3 and DNA methylation. Hence Mll2, which is orthologous to Drosophila Trithorax, is required to prevent Polycomb-Group repression of the Magoh2 promoter, and repression is further accompanied by DNA methylation. Early loss of Mll2 in utero recapitulated the embryonic lethality found in Mll2-/- embryos. However, loss of Mll2 after E11.5 produced mice without notable pathologies. Hence Mll2 is not required for late development, stem cells or homeostasis in somatic cell types. However it is required in the germ cell lineage. Spermatogenesis was lost upon removal of Mll2, although spermatogonia A persisted. Conclusion These data suggest a bimodal recruit and maintain model whereby Mll2 is required to establish certain epigenetic decisions during differentiation, which are then maintained by redundant mechanisms. We also suggest that these mechanisms relate to the epigenetic maintenance of CpG island promoters.

Hest shock protein 70 (Hsp70) class of chaperones is highly conserved and present ubiquitously in all cellular organisms They play important role in folding of nascent polypeptides and translocation of precursor proteins to endoplasmic reticulum, mitochondria and chloroplast Hsp70 assists in assembly of proteins complexes as well as in disassembly e g uncoatmg of clathrin coated vesicles Chaperone function of Hsp70 is modulated by cochaperones of DnaJ class, Hip, Hop etc Hsp70 is a component of multi chaperone complex with Hsp90 and helps in maturation of kinases or transcription factors. Plasmodium falciparum is responsible for most severe form of human malaria Plasmodmm in its intraerythrocytic cycle presents an example of a cell with multiple, complex membrane bound structures both inside the parasite as well as m the infected erythrocyte cytosol Parasite deploys proteins in host erythrocyte cytosol, at erythrocyte plasma membrane or traffics them for secretion outside the infected cell in addition to trafficking of proteins to its own organelles like mitochondria, apicoplast, food vacuole, ER etc It is of interest to malaria biologists to understand these trafficking events and role of chaperones in regulating them This study was aimed at understanding the function(s) of Hsp70 in Plasmodium infected erythrocyte in protein maturation and trafficking events We have attempted to study Hsp70 chaperone present in Plasmodium infected erythrocytes We have largely focused on the cytosohc Hsp70, PfHsp70, in the parasite and systematically analyzed its expression, localization, abundance and complexes in the intraerythrocytic cycle To gain insight into its function, we have identified a subset of PfHsp70 interacting proteins, parasite Hsp90, Hsp70-3, Hsp60 and beta tubulin by coimmunoprecipitation experiments in conjunction with proteomic tools like 2DGE and mass spectrometry Parasite Hsp60 is a mitochondria-targeted protein and we have examined the involvement of PfHsp70 in translocation of Hsp60 precursor protein to parasite mitochondrion PfHsp70 and PfHsp90 were found to be present in a complex Geldanarnycm, a drug that affects Hsp70-Hsp90 complex, was used to investigate the role of PfHsp70 in parasite protein trafficking Since there are no known parasite derived chaperones in erythrocyte cytosol compartment, we have examined the possible "involvement of host Hsp70 in supporting transport and assembly of parasite proteins in erythrocyte cytosol Hsp70 in Plasmodium falciparum intraerythrocytic cycle P. falciparum genome codes for five Hsp70 homologs Two of these, pfHsp70-l and PfBiP are expressed in intraerythrocytic stage and have been localized to nucleocytoplasmic and endoplasmic reticulum fraction of the parasite respectively We have focused this study on PfHsp70 of the parasite We show that PfHsp70 is an abundant protein in the cytosol constituting about 2% of the total soluble pool It gets further induced during stress like heat shock and translocates to nuclear fraction indicating that PfHsp70 may be involved in protective function in the parasite nucleus during stress Nuclear translocation of mammalian Hsp70 during stress has been linked to its phosphorylation at Tyr524 We found PfHsp70 to be phosphorylated by in vivo phosphate labeling m the parasite Analysis of PfHsp70 by 2-dimensional gel electrophoresis on narrow gradient IPG strips indicated that it exists in four forms differing in their isoelectnc points (pi) Phosphatase treatment combined with analysis using a phosphorylation prediction tool,Proteomod (http //www biochem use ernet in/proteomod html) suggested that PfHsp70 is phosphorylated at three residues in the parasite The extent of phosphorylation of PfHsp70 may determine substrate specificity or subcellular localization or both Using 2DGE and mass spectrometry approach, we also identified chaperones like Hsp909 BiP, Hsp60, and protein disulphide isomerase (PDI) m P falciparum proteome In summary, PfHsp70 appears to be a highly abundant, cytosohc chaperone It is inducible by stress and multiply phosphorylated and is likely to participate in multiple processes in the parasite. PfHsp70 complexes and interacting proteins in the parasite To gam insight into the functions of Hsp70, we looked for PfHsp70 interacting proteins in the parasite We used gel filtration chromatography to resolve and enrich PfHsp70 complexes and also employed coimmunoprecipitation approach to identify interacting proteins We found parasite Hsp90, Hsp70-3, Hsp60 and beta-tubulin interact with PfHsp70 Fractionation of parasite lysate indicated that PfHsp70 is present in two major complexes of 200 kDa and 450 kDa We find that PfHsp90 interacts with PfHsp70 and both are present in 450 kDa complex Our analysis indicated that 450-kDa complex is like Hsp70-Hsp90 multichaperone complex described in mammalian cells while 200 kDa complex is likely to be an Hsp70-cochaperone complex Smaller complex appears to be a precursor for multichaperone complex Use of an Hsp90 inhibitor, geldanamycin (GA), to study the function of this multi chaperone showed that GA inhibits parasite growth Maturation of four phosphoproteins interacting with PfHsp70 was affected by GA implicating them in regulation of parasite growth GA appeared to mediate its effects by inhibiting H§p^0 phosphorylation Amongst the other three interacting proteins, PfHsp70-3 is amoveJ/Hsp70 homolog that was found at the protein level for the first time in this study PfHsp60 is mitochondria-targeted protein in the parasite and it is likely that cytoshc PfHsp70 helps in translocation of PfHsp60 to mitochondria from cytosol Tubuhn is a cytoskeletal protein and its interaction with PfHsp70 suggests possible role of PfHsp70 in cytoskeleton organization during invasion, growth or cell division In all, we find that Hsp70 in the parasite exist in a multi chaperone complex with Hsp90 which might be responsible for maturation of signaling molecules important for growth The smaller complex of PfHsp70 is a precursor of multi chaperone complex and is likely to be an Hsp70- co chaperone complex Role of Hsp70 in protein translocation and trafficking Cytosolic Hsp70 aids in translocation of precursor proteins from cytosol to mitochondria (or chloroplast) We found a mitochondnal chaperone, PfHsp60, interact with PfHsp70 and we examined the possibility that PfHsp60 translocation is assisted by cytosolic PfHsp70 We found that PfHsp60 had a cleavable, N-thermal targeting sequence Examination of PfHsp60 forms present in mitochondnal and cytosolic fraction of the parasite showed that mitochondnal form was more acidic in pi than cytosolic form as expected after targeting sequence cleavage Cytosolic PfHsp60 interacted with both PfHsp70 and PfHsp90 Interestingly, while mitochondnal PfHsp60 appeared to be in a chaperonm like complex, as expected, cytosolic form was present in smaller ohgomeric complex of about 450 kDa This suggested that PfHsp60 precursor form could be bound to multichperone complex All these experiments together strongly indicated that PfHsp60 precursor interacts with cytosolic Hsp70 and Hsp90 before former's translocation into mitochondria This interaction might be required to keep the precursor in the transport competent state P falciparum lives inside a vacuole in the infected cells but it deploys a number of proteins to host cell cytosol and to the plasma membrane To examine the involvement of multichaperone complex in trafficking, we studied the effect of GA on targeting of two parasite proteins, knob associated histidme-rich protein (KAHRP) and glycogen synthase kinase (GSK) KAHRP is indispensable for the formation of cytoadherence complexes called knobs at erythrocyte plasma membrane We found that KAHRP transport to erythrocyte plasma membrane was blocked in GA-treated parasites and it appeared all over the infected cell Further analysis showed that GA caused block in KAHRP transport at some step beyond its exit from parasite ER The targeting of GSK to membranous inclusions in the infected RBC cytosol was not severely affected m the GA-treated parasites suggesting that GSK transport may not be regulated by multi chaperone complex It also indicated that parasite may be using different pathways for trafficking of proteins to the host compartment In summary, PfHsp70 and PfHsp90 interact with PfHsp60 precursor in the cytosol They probably help keep the precursor in transport competent form before arrival at the translocase complex of mitochondria The multi chaperone complex may also be important for trafficking of at least one parasite protein, KAHRP, to the host cell compartment Analysis of erythrocyte Hsp70 in Plasmodium falciparum infected cells The remodeled plasma membrane of parasite-infected erythrocytes is important for the cytoadherence property of the infected cells Knobs, supramolecular complexes on the infected cell surface, formed by parasite proteins, PfEMPl, KAHRP, and PfEMP3 are responsible for cytoadherence of infected cells to vascular endothehum or placenta KAHRP transport is BFA-sensitive inside the parasite while PfEMP proteins undergo vesicle mediated trafficking in the erythrocyte cytosol The involvement of molecular chaperones has been implicated in the trafficking and assembly of knob components in the erythrocyte cytosol There is no evidence for the presence of bona fide parasite derived chaperones in the host compartment The chaperones of the erythrocyte origin, Hsp70, Hsp90, Hip and Hop were readily detected in the host cytosol, on the other hand By analyzing localization, abundance and biochemical characteristics of the host chaperones of erythrocyte origin, we examined if host chaperones are being utilized by the parasite for its functions Localization experiment showed that while PfHsp70, PfHsp90 and PfBiP were present in the parasite compartment, host-Hsp70 was present in erythrocyte cytosol fraction Host~Hsp70 was about 60% as abundant as PfHsp70 and was potentially capable of facilitating chaperone function in the erythrocyte cytosol Though host-Hsp70 was soluble in unmfected cells, it was present in membrane bound, triton-insoluble complexes, containing KAHRP, in infected cells Since knobs are triton-insoluble complexes at the erythrocyte plasma membrane, we isolated erythrocyte ghost (plasma membrane) fraction and could detect both Hsp70 and KAHRP Hsp70 association with erythrocyte plasma membrane was specific as it could be crosshnked to KAHRP in ghost fraction of infected cells Host-hsp70 was present in purified cytoskeleton fraction containing knobs from infected cells along with cochaperone Hop All these evidences suggest that parasite may be exploiting host-Hsp70 in erythrocyte cytosol compartment Summary This study gives insight into some functions performed by PfHsp70 in mtraerythrocytic cycle of malarial parasite PfHsp70 is an abundant cytosohc chaperone in the parasite It gets induced during stress and translocates to the nucleus It is also phosphorylated at three sites Analysis of Pfhsp70 complexes shows that it is present in bimodal complexes (450 kDa and 200 kDa), which are in equilibrium PfHsp70 and PfHsp90 interact and are part of 450 kDa multichaperone complex This multichaperone complex appears to regulate trafficking of one parasite protein to host cytosol compartment In addition, PfHsp70 and PfHsp90 are also bound to mitochondria-targeted PfHsp60 precursor in the cytosol probably keeping them m a transport competent state In addition to PfHsp90 and PfHsp60, PfHsp70 interacts with a novel Hsp70 homolog of the parasite, PfHsp70-3, and cytoskeletal protein, beta-tubuhn Examination of chaperones available in erythrocyte cytosol, showed that parasite chaperones were absent while host chaperone (Hsp70) was present and exhibited altered properties during parasite infection It was associated with membrane-bound, triton-insoluble complexes on the infected cell plasma membrane suggesting that host-Hsp70 might be involved in trafficking and/or assembly of parasite proteins In all, PfHsp70, as part of multichaperone complex, appears to be regulating translocation and trafficking of parasite proteins to organellar locations or outside the parasite Host-Hsp70, in erythrocyte cytosol, might also be engaged in specific chaperone function upon infection

Background Histone methylation is thought to be central to the epigenetic mechanisms that maintain and confine cellular identity in multi-cellular organisms. To examine epigenetic roles in cellular homeostasis, we conditionally mutated the histone 3 lysine 4 methyltransferase, Mll2, in embryonic stem (ES) cells, during development and in adult mice using tamoxifen-induced Cre recombination. Results In ES cells, expression profiling unexpectedly revealed that only one gene, Magoh2, is dependent upon Mll2 and few other genes were affected. Loss of Mll2 caused loss of H3K4me3 at the Magoh2 promoter and concomitant gain of H3K27me3 and DNA methylation. Hence Mll2, which is orthologous to Drosophila Trithorax, is required to prevent Polycomb-Group repression of the Magoh2 promoter, and repression is further accompanied by DNA methylation. Early loss of Mll2 in utero recapitulated the embryonic lethality found in Mll2-/- embryos. However, loss of Mll2 after E11.5 produced mice without notable pathologies. Hence Mll2 is not required for late development, stem cells or homeostasis in somatic cell types. However it is required in the germ cell lineage. Spermatogenesis was lost upon removal of Mll2, although spermatogonia A persisted. Conclusion These data suggest a bimodal recruit and maintain model whereby Mll2 is required to establish certain epigenetic decisions during differentiation, which are then maintained by redundant mechanisms. We also suggest that these mechanisms relate to the epigenetic maintenance of CpG island promoters.