Mll4: Key Functions in Chromatin Regulation and Development

MLL4 (also known as KMT2D) is a histone methyltransferase that modifies chromatin structure to regulate gene expression. As part of the COMPASS family, it influences transcription through histone H3 lysine 4 (H3K4) methylation, affecting gene activation and cellular differentiation. This function is essential for normal development and tissue homeostasis.

MLL4 interacts with other chromatin modifiers and signaling pathways to fine-tune developmental processes. Understanding its role provides insight into how epigenetic mechanisms contribute to health and disease.

Chromatin Organization And Regulation

MLL4 (KMT2D) plays a central role in chromatin structure regulation, shaping its dynamic landscape. Chromatin consists of nucleosomes—histone proteins wrapped with DNA—that control gene accessibility. Histone modifications either promote or restrict transcription. MLL4 catalyzes the methylation of histone H3 at lysine 4 (H3K4), a mark associated with active transcription. This function helps establish and maintain enhancer elements, critical for precise gene regulation during development.

Enhancers are DNA sequences that interact with promoters to drive transcription, and their activity is tightly linked to chromatin modifications. MLL4 is found at enhancers marked by H3K4 monomethylation (H3K4me1), a signature of poised or active regulatory elements. Chromatin immunoprecipitation sequencing (ChIP-seq) studies show that MLL4 occupancy correlates with enhancer activation in various cell types, including embryonic stem cells. Transcription factors and co-regulatory proteins recruit MLL4 to these regions, ensuring gene expression patterns respond to developmental and environmental cues.

Beyond enhancer regulation, MLL4 facilitates interactions between distant genomic regions. Chromosome conformation capture techniques, such as Hi-C and 4C-seq, reveal that MLL4-regulated enhancers frequently contact target gene promoters. These interactions, stabilized by chromatin architectural proteins like cohesin and CTCF, help establish topologically associating domains (TADs). Disruptions in MLL4 function can lead to misregulated gene expression and developmental defects.

Molecular Architecture

The structural composition of MLL4 (KMT2D) enables its function as a histone methyltransferase, orchestrating chromatin modifications. As a COMPASS family member, MLL4 has distinct domains that mediate interactions with chromatin, cofactors, and regulatory proteins. Its catalytic SET domain, located at the C-terminal region, transfers methyl groups to histone H3 lysine 4 (H3K4), a modification linked to active enhancers. Although homologous to other KMT2 family members, MLL4’s specificity is dictated by its structural features and interacting partners.

Adjacent to the SET domain, MLL4 contains regions that bind cofactors, including the Win (WDR5 interaction) motif, which stabilizes its enzymatic function. Structural studies using cryo-electron microscopy and X-ray crystallography show that the Win motif engages WDR5, bridging MLL4 with COMPASS components such as ASH2L and RBBP5, which enhance methyltransferase efficiency. Disruptions in these interactions impair MLL4 function, leading to epigenetic dysregulation.

Additional domains contribute to chromatin targeting and enhancer specificity. PHD (Plant Homeodomain) fingers recognize methylated histones, reinforcing MLL4’s recruitment to active regulatory regions, while FY-rich (FYRN and FYRC) domains stabilize enhancer-promoter communication. These structural elements enable MLL4 to bridge distal enhancers with their target genes, fine-tuning transcriptional output.

Biochemical Mechanisms

MLL4 (KMT2D) regulates gene expression by recognizing specific histone substrates, catalyzing methylation reactions, and establishing distinct epigenetic marks. These processes are controlled by cofactor interactions and chromatin context, ensuring MLL4 activity is directed toward appropriate genomic regions.

Substrate Recognition

MLL4 specifically targets histone H3 at lysine 4 (H3K4), a key residue in transcriptional regulation. The SET domain forms a catalytic pocket that accommodates the H3 tail. Structural analyses show that MLL4 interacts with nucleosomes rather than free histone peptides, indicating that chromatin context influences its activity. The presence of additional histone modifications, such as H3K27 acetylation (H3K27ac), enhances MLL4 recruitment to active enhancers. Transcription factors and chromatin-associated proteins help guide MLL4 to its target sites, ensuring its activity is restricted to regulatory regions that require precise epigenetic modulation.

Enzymatic Activity

MLL4’s SET domain transfers methyl groups from S-adenosylmethionine (SAM) to H3K4. Unlike other KMT2 family members that generate di- and trimethylation marks, MLL4 primarily catalyzes monomethylation (H3K4me1), a modification enriched at enhancers. This enzymatic preference is dictated by structural constraints within the SET domain. COMPASS complex components such as ASH2L and RBBP5 enhance substrate binding and catalytic turnover. Mutations affecting the SET domain or cofactor interactions impair MLL4 activity, disrupting enhancer function and transcriptional regulation.

Methylation Patterns

MLL4-dependent H3K4me1 marks define enhancer regions, distinguishing them from promoters marked by H3K4me3. Genome-wide studies show that these marks facilitate enhancer-promoter communication. Chromatin remodeling factors and histone demethylases fine-tune enhancer activity in response to developmental and environmental cues. MLL4 also cooperates with histone acetyltransferases such as CBP/p300, which deposit H3K27ac to reinforce active enhancer states. This combinatorial modification pattern ensures precise gene expression programs. Disruptions in these methylation dynamics contribute to developmental disorders and disease.

Cross-Talk With Other Regulatory Proteins

MLL4 (KMT2D) functions within a network of chromatin regulators, ensuring precise gene expression. Its activity at enhancers is influenced by interactions with proteins that modulate chromatin accessibility, transcription factor recruitment, and epigenetic stability.

A key interaction involves CBP/p300, histone acetyltransferases that deposit H3K27ac, a mark of active enhancers. MLL4 and CBP/p300 co-occupy enhancer elements, reinforcing each other’s activity—MLL4-mediated H3K4 monomethylation provides a foundation for acetylation, while CBP/p300 activity stabilizes enhancer function. This interplay ensures enhancers remain accessible for transcription factor binding during differentiation. MLL4 also interacts with chromatin remodeling complexes such as SWI/SNF, which reposition nucleosomes to facilitate transcriptional activation. The ATP-dependent activity of SWI/SNF enhances MLL4 function by exposing enhancer elements.

Developmental Significance

MLL4 (KMT2D) is essential for embryonic development, regulating gene expression programs that drive cellular differentiation and organogenesis. Knockout studies in model organisms, including mice and zebrafish, show that loss of MLL4 leads to severe developmental abnormalities, including defects in neural, cardiac, and skeletal formation. These findings highlight its necessity in orchestrating complex developmental pathways.

Beyond early development, MLL4 continues to regulate gene expression in postnatal development and tissue maintenance. In stem and progenitor cells, it maintains enhancer activity for genes involved in self-renewal and differentiation. Conditional knockout models reveal that MLL4 deficiency in specific tissues, such as the brain or heart, results in structural and functional defects. In neural development, MLL4 activates genes that guide neuronal migration and synaptic maturation. In cardiac development, it regulates enhancer elements controlling heart morphogenesis and contractility. These roles underscore how MLL4-mediated chromatin modifications ensure developmental precision.

Links To Health Conditions

Mutations in MLL4 (KMT2D) are linked to developmental disorders and disease. One of the most well-characterized conditions is Kabuki syndrome, a congenital disorder marked by intellectual disability, growth deficiencies, and distinctive facial features. Loss-of-function mutations in MLL4 disrupt enhancer activation, leading to widespread gene misregulation. Studies using patient-derived cells show that MLL4 mutations impair H3K4 monomethylation, reducing enhancer activation potential. This epigenetic dysregulation contributes to the phenotypic variability observed in Kabuki syndrome.

MLL4 mutations are also implicated in cancers, particularly lymphoma and hepatocellular carcinoma. As a tumor suppressor, MLL4 helps maintain enhancer landscapes that regulate cell proliferation and differentiation. Inactivating mutations lead to enhancer reprogramming, allowing oncogenic pathways to become aberrantly activated. Genome-wide cancer analyses reveal that MLL4-deficient tumors exhibit widespread enhancer deregulation, driving uncontrolled cell growth. Restoring MLL4 function in cancer models partially reverses malignant phenotypes, suggesting potential therapeutic strategies targeting its epigenetic activity.