Recombinant Chicken Putative Polycomb group protein ASXL2 (ASXL2), partial

What is ASXL2 and what are its known functions?

ASXL2 (Additional Sex Combs Like 2) is a putative Polycomb group protein that functions as a transcriptional regulator. It is a mammalian homologue of the Drosophila gene ASX, which encodes an Enhancer of Trithorax and Polycomb (ETP) protein that regulates histone methylation . ASXL2 has been demonstrated to play essential roles in several biological processes including:

• Hematopoietic stem cell self-renewal and normal blood cell development

• Regulation of glucose and lipid metabolism through interaction with PPARγ

• Skeletal homeostasis and bone mineral density regulation

• Tumor suppression in certain leukemia contexts, particularly in AML1-ETO-driven acute myeloid leukemia

ASXL2 exerts these functions primarily through epigenetic mechanisms, influencing gene expression patterns by modulating chromatin accessibility and structure at key genomic loci.

How does ASXL2 differ from other ASXL family members?

While ASXL1 has been extensively studied due to its frequent mutations in leukemia, ASXL2 has distinct, non-overlapping functions. Research has demonstrated that:

• ASXL2 deletion affects a much larger set of genes (2,986) compared to ASXL1 deletion (129) in hematopoietic stem and progenitor cells

• The transcriptional effects of ASXL2 and ASXL1 show minimal overlap, indicating they regulate different gene sets

• ASXL2 target genes significantly overlap with those regulated by AML1-ETO and RUNX1, which is not observed with ASXL1-regulated genes

• While both are implicated in leukemogenesis, ASXL2 mutations are particularly associated with AML cases bearing the RUNX1-RUNX1T1 (AML1-ETO) fusion

These differences highlight the specialized and non-redundant roles of ASXL family members in cellular function and disease pathogenesis.

What expression patterns and tissue specificity does ASXL2 exhibit?

ASXL2 shows varied expression across tissues, with particularly important functions in:

• Hematopoietic cells, especially within the myeloid lineage and hematopoietic stem cells

• Osteoclasts and cells involved in bone remodeling

• Metabolic tissues including liver and muscle, where it influences insulin signaling

Gene cluster analysis has revealed that genes most prominently co-expressed with ASXL2 are those mediating myeloid differentiation, suggesting a significant role in monocyte/macrophage lineage development and by extension, osteoclast function .

How does ASXL2 regulate normal hematopoietic stem cell function?

ASXL2 is essential for normal hematopoietic stem cell (HSC) self-renewal and maintenance. Experimental evidence demonstrates:

• Complete deletion of Asxl2 in mouse models results in rapid leukopenia and thrombocytopenia, indicating critical requirements for blood cell production

• In competitive transplantation assays, Asxl2-deficient HSCs exhibit profound defects in self-renewal capacity and reconstitution ability

• Even heterozygous loss of Asxl2 causes substantial decrements in HSPC self-renewal, demonstrating haploinsufficiency for normal stem cell function

• Asxl2 deletion affects HSPCs more severely than mature circulating blood cells, suggesting a primary role in stem/progenitor cell biology

The molecular mechanisms underlying these effects likely involve ASXL2's regulation of chromatin state at key hematopoietic loci, influencing transcriptional programs essential for stem cell maintenance.

What is the paradoxical role of ASXL2 in leukemogenesis?

ASXL2 exhibits a complex, context-dependent function in leukemia development:

• ASXL2 normally functions as a haploinsufficient tumor suppressor, as its loss promotes leukemogenesis in the context of AML1-ETO expression

• Despite being required for normal hematopoiesis, Asxl2 loss specifically cooperates with the AML1-ETO fusion protein to accelerate leukemia development

• Mutations in ASXL2 occur as heterozygous alterations in leukemia patients, consistent with the experimental finding that deletion of even a single copy of Asxl2 can promote leukemogenic effects

• ASXL2 mutations (such as p.T740NfsX16 and p.E1287X) result in decreased protein stability, suggesting they are primarily loss-of-function alterations

This paradoxical relationship where ASXL2 is both required for normal hematopoiesis yet its reduction can promote leukemia in specific genetic contexts illustrates the complex role of epigenetic regulators in cancer.

How do ASXL2 mutations specifically cooperate with AML1-ETO in leukemogenesis?

The specific cooperation between ASXL2 mutations and AML1-ETO in leukemia development involves several molecular mechanisms:

• ASXL2 target genes strongly overlap with those regulated by RUNX1 and AML1-ETO, creating a mechanistic basis for their functional interaction

• Loss of ASXL2 alters chromatin accessibility at putative enhancers of key leukemogenic loci in AML1-ETO-expressing cells

• Genes differentially expressed following Asxl2 loss significantly overlap with genes upregulated by the AML1-ETO oncoprotein

• ASXL2 likely influences the epigenetic landscape at AML1-ETO binding sites, modulating the oncogenic transcriptional program driven by this fusion protein

This relationship explains the clinical observation that ASXL2 mutations frequently occur in AML patients bearing the RUNX1-RUNX1T1 (AML1-ETO) fusion.

How does ASXL2 influence glucose homeostasis and insulin sensitivity?

ASXL2 plays a critical role in metabolic regulation, particularly glucose homeostasis:

• ASXL2−/− mice exhibit glucose intolerance and insulin resistance

• These mice show elevated basal serum glucose in both fasting and fed states despite normal circulating insulin levels, typical of early-onset Type 2 diabetes

• Asxl2-deficient mice demonstrate attenuated clearance of glucose following insulin challenge

• At the molecular level, liver and muscle tissues from ASXL2-deficient mice fail to phosphorylate Akt or insulin receptor in response to insulin administration

The mechanism appears to involve ASXL2's interaction with PPARγ, a master regulator of insulin sensitivity. ASXL2 activates PPARγ in multiple cellular contexts, and its absence impairs PPARγ-mediated metabolic regulation.

What is the role of ASXL2 in bone mineral density regulation and skeletal homeostasis?

Genome-wide screening has identified ASXL2 as a significant regulator of bone mineral density (BMD):

• SNPs within ASXL2 are associated with BMD in multiple human cohorts, establishing clinical relevance

• ASXL2−/− mice exhibit increased trabecular bone mass (over 400%) due to impaired osteoclast function

• ASXL2 is highly co-expressed with genes mediating myeloid differentiation, which includes the monocyte/macrophage lineage that gives rise to osteoclasts

• ASXL2 deficiency results in attenuated osteoclastogenesis both in vitro and in vivo

These findings position ASXL2 as an important regulator of bone remodeling, primarily through its effects on osteoclast development and function.

What are the optimal experimental approaches for studying ASXL2 function in vitro?

Several methodological approaches have proven effective for investigating ASXL2 function:

Expression Systems:

• E. coli, yeast, baculovirus, or mammalian cell expression systems can all be used for recombinant ASXL2 production, with purities ≥85% as determined by SDS-PAGE

• Mammalian expression vectors encoding full-length wild-type ASXL2 (1–1435) or disease-associated mutants (e.g., p.T740NfsX16 and p.E1287X) can be employed to study protein stability and function

Functional Analysis:

• Protein stability assessments using cycloheximide chase assays have revealed reduced stability of mutant ASXL2 relative to wild-type protein

• ChIP-seq approaches can identify genomic binding sites and chromatin modifications influenced by ASXL2

• RNA-seq of purified cell populations (e.g., lineage-negative Sca−1+ c-Kit+ [LSK] cells) allows comprehensive analysis of ASXL2's transcriptional effects

Antibody-Based Approaches:

• Multiple antibody options exist for ASXL2 detection, including monoclonal and polyclonal antibodies applicable for Western blotting, ELISA, and immunohistochemistry

• Protein purification techniques like antigen affinity purification yield high-quality antibodies for research applications

What genetic models are available for studying ASXL2 function in vivo?

Several genetic models have been developed to investigate ASXL2 function:

Constitutive Knockout:

• Complete constitutive deletion of Asxl2 is associated with substantial perinatal lethality, limiting its research utility

Conditional Knockout:

• A conditional allele with LoxP sites flanking exon 11 of Asxl2 has been generated

• This model can be crossed with tissue-specific or inducible Cre lines, such as Mx1-cre for inducible deletion in hematopoietic tissues

• The conditional system allows post-natal deletion, circumventing developmental lethality issues

Verification Methodologies:

• Western blotting and quantitative PCR with reverse transcription of bone marrow mononuclear cells can confirm Asxl2 deletion efficiency

• Functional assays including competitive transplantation experiments assess the consequences of Asxl2 deletion on stem cell function

What approaches can be used to investigate ASXL2 in human disease contexts?

Multiple approaches facilitate the study of ASXL2 in human disease:

Genetic Analysis:

• Genome-wide association studies have identified ASXL2 SNPs associated with bone mineral density in human populations

• Targeted sequencing of ASXL2 in patient samples, particularly from AML1-ETO-positive AML cases, can identify disease-associated mutations

Expression Analysis:

• Human ASXL2 cDNA clones are available for expression studies

• ASXL2 antibodies reactive to human protein enable detection in patient samples

Functional Studies:

• Expression of wild-type versus mutant ASXL2 in human cell lines allows assessment of protein stability and function

• siRNA-mediated knockdown can model ASXL2 deficiency in human cells

How do chicken, mouse, and human ASXL2 proteins compare in structure and function?

Comparative analysis reveals considerable conservation across species:

The high degree of conservation, particularly in functional domains, suggests fundamental roles for ASXL2 that have been maintained throughout vertebrate evolution. This conservation facilitates translational research using model organisms.

How does ASXL2 function in epigenetic regulation compare to other chromatin modifiers?

ASXL2 possesses distinctive features compared to other chromatin modifiers:

• As an ETP (Enhancer of Trithorax and Polycomb) protein, ASXL2 can potentially modulate both activating and repressive chromatin marks, unlike proteins dedicated to single modifications

• ASXL2 influences chromatin accessibility at enhancers of key regulatory loci, suggesting a role in modulating enhancer-promoter interactions

• Unlike many chromatin modifiers with broad effects, ASXL2 demonstrates context-specific functions, as evidenced by its particular relevance in AML1-ETO-driven leukemia

• ASXL2's effects on transcription (2,986 differentially expressed genes upon deletion) are much more extensive than those of the related ASXL1 (129 genes), despite both being chromatin-associated proteins

This distinctive profile positions ASXL2 as a specialized epigenetic regulator with context-dependent functions rather than a general chromatin modifier.