Our studies establish LEDGF as a crucial cofactor required for both the oncogenic and tumor suppressor functions of MLL/menin complexes. LEDGF interacts conjointly with MLL and menin on the chromatin of cancer-associated genes to mediate MLL-dependent transcription pathways (). In this context, menin serves as an adaptor to link MLL with LEDGF. A subset of menin mutations in MEN1 tumors is particularly informative as they abrogate interactions with LEDGF, but not MLL, yet compromise MLL/menin activity. Genetic evidence that MLL functions with LEDGF in its normal developmental role to regulate HOX
gene expression during establishment of the embryonic body plan is suggested by the phenotypic overlaps of Mll
- and Ledgf
-deficient mice, which both display skeletal malformations representative of anterior and posterior homeotic transformations (Sutherland et al., 2006
; Yu et al., 1995
). Thus, LEDGF is an essential component of the MLL/menin HMT complex in the setting of its normal and pathologic activities.
Model for the role of LEDGF in the normal and neoplastic functions of the MLL/menin HMT complex
LEDGF has previously been implicated in various transcriptional processes and cellular functions. Originally discovered based on its co-fractionation with the general transcriptional co-activator PC4, LEDGF reportedly associates with transcriptional activators and components of the basal transcriptional machinery including RNA pol II subunits (Ge et al., 1998
), and contributes to the transcriptional response following environmental stress (Shinohara et al., 2002
). Coincidently, menin is also a regulator of stress-induced response in fruit flies, which transcriptionally up-regulate expression of various heat shock proteins following stressful stimuli (Papaconstantinou et al., 2005
). Menin has been reported to co-IP RNA pol II (Hughes et al., 2004
), raising the possibility that it links MLL with LEDGF/p75 in higher-order complexes of dynamic composition to regulate specific stages of transcription.
In addition to its transcriptional role, LEDGF is important for lentiviral integration (Ciuffi et al., 2005
; Llano et al., 2006
; Shun et al., 2007
). LEDGF is the dominant binding partner for HIV-1 integrase in human cells (Cherepanov et al., 2003
), and tethers it to host chromosomes (Maertens et al., 2003
), thereby serving a major role in determining the highly distinctive pattern of lentiviral genome integration within active transcription units (Ciuffi and Bushman, 2006
). Thus, physical association of LEDGF with MLL/menin on chromatin may provide a molecular basis for the selective integration of HIV-1 into actively transcribed regions since the epigenetic mark placed by MLL is involved in maintaining chromatin in a state conducive for transcription (Li et al., 2007
). A tethering role for LEDGF likely extends to other host proteins as well since the chromosomal association of JPO2, a MYC-interacting protein with transforming activity, is also strictly dependent on LEDGF (Maertens et al., 2006
). It remains to be determined if these various interactions, which target the IBD of LEDGF, are mutually exclusive with MLL/menin and what their implications may be for anti-lentiviral therapy.
The association of LEDGF with chromatinized DNA is critically dependent on its PWWP domain (Botbol et al., 2008
). This highly conserved motif is present in a variety of chromatin-associated proteins involved in transcriptional regulation, DNA repair and methylation (Stec et al., 2000
). It has structural similarities with Tudor, chromo and MBT domains, all of which are implicated in binding methylated Lys residues on histones (Maurer-Stroh et al., 2003
; Li et al. 2007
). Structural similarities with the ligand binding cavities of these evolutionally related domains strongly suggest that the PWWP domain binds to a currently undefined component of chromatin, although a possible role in non-specific DNA binding has been suggested as well (Lukasik et al., 2006
; Nameki et al., 2005
; Sue et al., 2004
; Qiu et al., 2002
). Compelling evidence for the role of LEDGF in targeting the MLL/menin HMT complex to chromatin is provided by grafting of its PWWP onto the MLL oncoprotein, which was fully capable of bypassing the requirement for menin in oncogenesis, Hox
gene mis-regulation and chromatin association. This artificial construct is structurally similar to the A. thaliana homologs of MLL (ATX1 and ATX2) (Alvarez-Venegas and Avramova, 2001
), which contain PWWP domains in their amino-terminal portions, providing evolutionary support for the functional link between MLL and LEDGF.
Our study indicates significant roles for LEDGF in menin-dependent growth control. Menin has been reported to potentially interact with DNA (La et al., 2004
) and several other proteins (Balogh et al., 2006
), however our data suggest that menin's sole role in MLL leukemia is to recruit LEDGF. Our observation that some MEN1-associated mutations specifically disrupt LEDGF binding and compromise MLL-dependent transcription also implicates LEDGF in MEN1 tumorigenesis. Accumulating evidence indicates that the physiologic growth responses of endocrine lineage cells are heavily dependent on the MLL/menin pathway through regulated expression of CDKIs (Franklin et al., 1998
; Milne et al., 2005
; Karnik et al., 2005
). In contrast to ATX proteins, the interaction of MLL with LEDGF critically mediated by menin is non-covalent, which provides a potential mechanism for regulating their conditional association. LEDGF is induced upon cellular stress stimuli including serum starvation (Huang et al., 2007
) and, interestingly, is secreted from and re-enters lens epithelial cells by penetrating the plasma membrane (Singh et al., 1999
). Thus, it is tempting to speculate that cell autonomous or non-autonomous induction of LEDGF may be implicated in the growth control of endocrine and other lineages, perhaps as part of a molecular switch for targeting of the MLL/menin HMT complex to chromatin ().
Our results provide a broader context for conceptualizing the various pathologies associated with LEDGF and its binding partners, which appear to be frequently targeted in diverse diseases. In addition to making essential contributions to MLL-mediated leukemias and endocrine tumorigenesis, LEDGF itself is targeted by chromosomal translocations in leukemia that result in its fusion with the nucleoporin NUP98 (Ahuja et al., 2000
). The molecular mechanism by which NUP98-LEDGF causes leukemia is unknown, however our data show that it is capable of associating with MLL/menin suggesting that it may also perturb the HOX pathway, compatible with the more frequent fusion of NUP98 with HOX proteins themselves in leukemias. Increased LEDGF expression is also a feature of some cancers including acute myeloid leukemia and tumors of breast and bladder origins (Daugaad et al., 2007; Huang et al., 2007
), whereas auto-antibodies to LEDGF are frequently present in patients with atopic dermatitis and other auto-immune disorders (Ganapathy and Casiano, 2004
). The specific roles of MLL/menin in these various diseases merit further investigation as they may have important implications for therapeutic interventions in cancer, auto-immunity and AIDS.