Elevated Id2 expression causes defective meiosis and spermatogenesis in mice.

BACKGROUND: Inhibitors of DNA binding (ID) proteins mainly inhibit gene expression and regulate cell fate decisions by interacting with E-proteins. All four ID proteins (ID1-4) are present in the testis, and ID4 has a particularly important role in spermatogonial stem cell fate determination. Several lines of evidence indicate that ID proteins are involved in meiosis; however, functional experiments have not been conducted to validate this observation. RESULTS: In this study, we report that ID2 is enriched in spermatocytes and that forced ID2 expression in germ cells causes defects in spermatogenesis. A detailed analysis demonstrated that Id2 overexpression (Id2 OE) decreased the total number of spermatogonia and changed the dynamics of meiosis progression. Specifically, spermatocytes were enriched in the zygotene stage, and the proportion of pachytene spermatocytes was significantly decreased, indicating defects in the zygotene-pachytene transition. The number of MLH1-positive foci per cell was decreased in pachytene spermatocytes from Id2 OE testes, suggesting abnormalities in recombination. Transcriptome analysis revealed that forced Id2 expression changed the expression of a list of genes mainly associated with meiosis and spermatid development. CONCLUSIONS: ID2 protein is expressed in spermatocytes, and its genetic ablation in the germline does not affect spermatogenesis, likely due to genetic compensation of its family members. However, forced Id2 expression changes meiosis progression and causes defects in spermiogenesis. These data provide important evidence that ID proteins play pivotal roles in male meiosis and spermatid development.

Inhibitor of DNA binding proteins revealed as orchestrators of steady state, stress and malignant hematopoiesis.

Adult mammalian hematopoiesis is a dynamic cellular process that provides a continuous supply of myeloid, lymphoid, erythroid/megakaryocyte cells for host survival. This process is sustained by regulating hematopoietic stem cells (HSCs) quiescence, proliferation and activation under homeostasis and stress, and regulating the proliferation and differentiation of downstream multipotent progenitor (MPP) and more committed progenitor cells. Inhibitor of DNA binding (ID) proteins are small helix-loop-helix (HLH) proteins that lack a basic (b) DNA binding domain present in other family members, and function as dominant-negative regulators of other bHLH proteins (E proteins) by inhibiting their transcriptional activity. ID proteins are required for normal T cell, B cell, NK and innate lymphoid cells, dendritic cell, and myeloid cell differentiation and development. However, recent evidence suggests that ID proteins are important regulators of normal and leukemic hematopoietic stem and progenitor cells (HSPCs). This chapter will review our current understanding of the function of ID proteins in HSPC development and highlight future areas of scientific investigation.

Transcriptional dynamics and epigenetic regulation of E and ID protein encoding genes during human T cell development.

T cells are generated from hematopoietic stem cells through a highly organized developmental process, in which stage-specific molecular events drive maturation towards αß and γδ T cells. Although many of the mechanisms that control αß- and γδ-lineage differentiation are shared between human and mouse, important differences have also been observed. Here, we studied the regulatory dynamics of the E and ID protein encoding genes during pediatric human T cell development by evaluating changes in chromatin accessibility, histone modifications and bulk and single cell gene expression. We profiled patterns of ID/E protein activity and identified up- and downstream regulators and targets, respectively. In addition, we compared transcription of E and ID protein encoding genes in human versus mouse to predict both shared and unique activities in these species, and in prenatal versus pediatric human T cell differentiation to identify regulatory changes during development. This analysis showed a putative involvement of TCF3/E2A in the development of γδ T cells. In contrast, in αß T cell precursors a pivotal pre-TCR-driven population with high ID gene expression and low predicted E protein activity was identified. Finally, in prenatal but not postnatal thymocytes, high HEB/TCF12 levels were found to counteract high ID levels to sustain thymic development. In summary, we uncovered novel insights in the regulation of E and ID proteins on a cross-species and cross-developmental level.

Signaling networks controlling ID and E protein activity in T cell differentiation and function.

E and inhibitor of DNA binding (ID) proteins are involved in various cellular developmental processes and effector activities in T cells. Recent findings indicate that E and ID proteins are not only responsible for regulating thymic T cell development but also modulate the differentiation, function, and fate of peripheral T cells in multiple immune compartments. Based on the well-established E and ID protein axis (E-ID axis), it has been recognized that ID proteins interfere with the dimerization of E proteins, thus restricting their transcriptional activities. Given this close molecular relationship, the extent of expression or stability of these two protein families can dynamically affect the expression of specific target genes involved in multiple aspects of T cell biology. Therefore, it is essential to understand the endogenous proteins or extrinsic signaling pathways that can influence the dynamics of the E-ID axis in a cell-specific and context-dependent manner. Here, we provide an overview of E and ID proteins and the functional outcomes of the E-ID axis in the activation and function of multiple peripheral T cell subsets, including effector and memory T cell populations. Further, we review the mechanisms by which endogenous proteins and signaling pathways alter the E-ID axis in various T cell subsets influencing T cell function and fate at steady-state and in pathological settings. A comprehensive understanding of the functions of E and ID proteins in T cell biology can be instrumental in T cell-specific targeting of the E-ID axis to develop novel therapeutic modalities in the context of autoimmunity and cancer.

Shifting gears: Id3 enables recruitment of E proteins to new targets during T cell development and differentiation.

Shifting levels of E proteins and Id factors are pivotal in T cell commitment and differentiation, both in the thymus and in the periphery. Id2 and Id3 are two different factors that prevent E proteins from binding to their target gene cis-regulatory sequences and inducing gene expression. Although they use the same mechanism to suppress E protein activity, Id2 and Id3 play very different roles in T cell development and CD4 T cell differentiation. Id2 imposes an irreversible choice in early T cell precursors between innate and adaptive lineages, which can be thought of as a railway switch that directs T cells down one path or another. By contrast, Id3 acts in a transient fashion downstream of extracellular signals such as T cell receptor (TCR) signaling. TCR-dependent Id3 upregulation results in the dislodging of E proteins from their target sites while chromatin remodeling occurs. After the cessation of Id3 expression, E proteins can reassemble in the context of a new genomic landscape and molecular context that allows induction of different E protein target genes. To describe this mode of action, we have developed the "Clutch" model of differentiation. In this model, Id3 upregulation in response to TCR signaling acts as a clutch that stops E protein activity ("clutch in") long enough to allow shifting of the genomic landscape into a different "gear", resulting in accessibility to different E protein target genes once Id3 decreases ("clutch out") and E proteins can form new complexes on the DNA. While TCR signal strength and cytokine signaling play a role in both peripheral and thymic lineage decisions, the remodeling of chromatin and E protein target genes appears to be more heavily influenced by the cytokine milieu in the periphery, whereas the outcome of Id3 activity during T cell development in the thymus appears to depend more on the TCR signal strength. Thus, while the Clutch model applies to both CD4 T cell differentiation and T cell developmental transitions within the thymus, changes in chromatin accessibility are modulated by biased inputs in these different environments. New emerging technologies should enable a better understanding of the molecular events that happen during these transitions, and how they fit into the gene regulatory networks that drive T cell development and differentiation.

KSHV RTA Induces Degradation of the Host Transcription Repressor ID2 To Promote the Viral Lytic Cycle.

The immediate early viral protein replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is essential for activating the lytic cycle of KSHV. RTA induces the KSHV lytic cycle by several mechanisms, acting as a viral transcription factor that directly induces viral and host genes and acting as a viral E3 ubiquitin ligase by degrading host proteins that block viral lytic replication. Recently, we have characterized the global gene expression changes in primary effusion lymphoma (PEL) upon lytic reactivation of KSHV, which also led to the identification of rapidly downregulated genes such as ID2, an inhibitor of basic helix-loop-helix transcription factors. Here, we demonstrate that ID2 overexpression in PEL ablates KSHV lytic reactivation, indicating that ID2 inhibits the KSHV lytic cycle. Furthermore, we show that while ID2 is highly expressed during latency, its protein level is rapidly reduced by 4 h postinduction during lytic reactivation. Our results indicate that RTA binds to ID2 and induces its degradation during the KSHV lytic cycle by N-terminal ubiquitination through the ubiquitin-proteasome pathway. Importantly, we found that not only KSHV RTA but also its Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68) homologs interact with ID2, and they can induce the degradation of all four members of the ID protein family, suggesting an evolutionarily conserved interplay between gammaherpesvirus RTAs and ID proteins. Taken together, we propose that ID2 acts as a repressor of the KSHV lytic cycle, which is counteracted by its RTA-mediated degradation. We also predict that ID proteins may act as restriction factors of the lytic phase of the other gammaherpesviruses as well. IMPORTANCE In addition to its transcription regulatory role, RTA is also known to have an E3 ubiquitin ligase activity, which RTA utilizes for inducing protein degradation. However, it is still largely unknown what host factors are downregulated during KSHV lytic reactivation by RTA-mediated protein degradation and what the biological significance of the degradation of these host factors is. In this study, we discovered that RTA employs N-terminal ubiquitination to induce degradation of ID2, a potent transcription repressor of host genes, via the ubiquitin-proteasome pathway to promote KSHV lytic reactivation in PEL cells. Furthermore, we found that not only KSHV RTA but also RTA of EBV and MHV68 gammaherpesviruses can induce the degradation of all four human ID proteins, indicating that the interplay between gammaherpesvirus RTAs and ID proteins is evolutionarily conserved.

ILC Differentiation in the Thymus.

The thymus provides a microenvironment conducive to the differentiation of innate lymphoid cells (ILCs), supplying IL-7 as well as Notch ligands. Early T cell precursors also express a number of obligatory transcription factors essential for ILC differentiation. Therefore, the thymus could be a powerhouse for ILC production. However, coordinated regulation by transcription factors and T cell receptor signaling events ensure that T cell production is the dominating output of the thymus. One group of the key regulators are the basic helix-loop-helix E protein transcription factors and their inhibitors, Id proteins. When E protein activities are downregulated, T cell development is blocked and massive ILC2 production occurs in the thymus. Normally, the thymus indeed generates a small number of ILCs, mostly group 2 ILCs (ILC2s). It has been shown in vitro that ILC2s can be differentiated from multipotent early T cell progenitors (ETPs) as well as committed T cell precursors. Moreover, thymus-derived ILC precursors have been found in the blood of adult mice. They then home to peripheral tissues and undergo differentiation into distinct ILC subsets. These ILC precursors may replenish tissue ILC pools in steady state or on demand in pathophysiological conditions. Collectively, emerging evidence suggests that the thymus plays an underappreciated role in ILC homeostasis.

Helix-Loop-Helix Proteins in Adaptive Immune Development.

The E/ID protein axis is instrumental for defining the developmental progression and functions of hematopoietic cells. The E proteins are dimeric transcription factors that activate gene expression programs and coordinate changes in chromatin organization. Id proteins are antagonists of E protein activity. Relative levels of E/Id proteins are modulated throughout hematopoietic development to enable the progression of hematopoietic stem cells into multiple adaptive and innate immune lineages including natural killer cells, B cells and T cells. In early progenitors, the E proteins promote commitment to the T and B cell lineages by orchestrating lineage specific programs of gene expression and regulating VDJ recombination of antigen receptor loci. In mature B cells, the E/Id protein axis functions to promote class switch recombination and somatic hypermutation. E protein activity further regulates differentiation into distinct CD4+ and CD8+ T cells subsets and instructs mature T cell immune responses. In this review, we discuss how the E/Id proteins define the adaptive immune system lineages, focusing on their role in directing developmental gene programs.

Homodimeric and Heterodimeric Interactions among Vertebrate Basic Helix-Loop-Helix Transcription Factors.

The basic helix-loop-helix transcription factor (bHLH TF) family is involved in tissue development, cell differentiation, and disease. These factors have transcriptionally positive, negative, and inactive functions by combining dimeric interactions among family members. The best known bHLH TFs are the E-protein homodimers and heterodimers with the tissue-specific TFs or ID proteins. These cooperative and dynamic interactions result in a complex transcriptional network that helps define the cell's fate. Here, the reported dimeric interactions of 67 vertebrate bHLH TFs with other family members are summarized in tables, including specifications of the experimental techniques that defined the dimers. The compilation of these extensive data underscores homodimers of tissue-specific bHLH TFs as a central part of the bHLH regulatory network, with relevant positive and negative transcriptional regulatory roles. Furthermore, some sequence-specific TFs can also form transcriptionally inactive heterodimers with each other. The function, classification, and developmental role for all vertebrate bHLH TFs in four major classes are detailed.

Id Proteins Promote a Cancer Stem Cell Phenotype in Mouse Models of Triple Negative Breast Cancer via Negative Regulation of Robo1.

Breast cancers display phenotypic and functional heterogeneity and several lines of evidence support the existence of cancer stem cells (CSCs) in certain breast cancers, a minor population of cells capable of tumor initiation and metastatic dissemination. Identifying factors that regulate the CSC phenotype is therefore important for developing strategies to treat metastatic disease. The Inhibitor of Differentiation Protein 1 (Id1) and its closely related family member Inhibitor of Differentiation 3 (Id3) (collectively termed Id) are expressed by a diversity of stem cells and are required for metastatic dissemination in experimental models of breast cancer. In this study, we show that ID1 is expressed in rare neoplastic cells within ER-negative breast cancers. To address the function of Id1 expressing cells within tumors, we developed independent murine models of Triple Negative Breast Cancer (TNBC) in which a genetic reporter permitted the prospective isolation of Id1+ cells. Id1+ cells are enriched for self-renewal in tumorsphere assays in vitro and for tumor initiation in vivo. Conversely, depletion of Id1 and Id3 in the 4T1 murine model of TNBC demonstrates that Id1/3 are required for cell proliferation and self-renewal in vitro, as well as primary tumor growth and metastatic colonization of the lung in vivo. Using combined bioinformatic analysis, we have defined a novel mechanism of Id protein function via negative regulation of the Roundabout Axon Guidance Receptor Homolog 1 (Robo1) leading to activation of a Myc transcriptional programme.

Id1 and Id3 Maintain Steady-State Hematopoiesis by Promoting Sinusoidal Endothelial Cell Survival and Regeneration.

Investigating mechanisms that regulate endothelial cell (EC) growth and survival is important for understanding EC homeostasis and how ECs maintain stem cell niches. We report here that targeted loss of Id genes in adult ECs results in dilated, leaky sinusoids and a pro-inflammatory state that increases in severity over time. Disruption in sinusoidal integrity leads to increased hematopoietic stem cell (HSC) proliferation, differentiation, migration, and exhaustion. Mechanistically, sinusoidal ECs (SECs) show increased apoptosis because of reduced Bcl2-family gene expression following Id gene ablation. Furthermore, Id1-/-Id3-/- SECs and upstream type H vessels show increased expression of cyclin-dependent kinase inhibitors p21 and p27 and impaired ability to proliferate, which is rescued by reducing E2-2 expression. Id1-/-Id3-/- mice do not survive sublethal irradiation because of impaired vessel regeneration and hematopoietic failure. Thus, Id genes are required for the survival and regeneration of BM SECs during homeostasis and stress to maintain HSC development.

A Small-Molecule Pan-Id Antagonist Inhibits Pathologic Ocular Neovascularization.

Id helix-loop-helix (HLH) proteins (Id1-4) bind E protein bHLH transcription factors, preventing them from forming active transcription complexes that drive changes in cell states. Id proteins are primarily expressed during development to inhibit differentiation, but they become re-expressed in adult tissues in diseases of the vasculature and cancer. We show that the genetic loss of Id1/Id3 reduces ocular neovascularization in mouse models of wet age-related macular degeneration (AMD) and retinopathy of prematurity (ROP). An in silico screen identifies AGX51, a small-molecule Id antagonist. AGX51 inhibits the Id1-E47 interaction, leading to ubiquitin-mediated degradation of Ids, cell growth arrest, and reduced viability. AGX51 is well-tolerated in mice and phenocopies the genetic loss of Id expression in AMD and ROP models by inhibiting retinal neovascularization. Thus, AGX51 is a first-in-class compound that antagonizes an interaction formerly considered undruggable and that may have utility in the management of multiple diseases.

Id4 promotes the elimination of the pro-activation factor Ascl1 to maintain quiescence of adult hippocampal stem cells.

Quiescence is essential for the long-term maintenance of adult stem cells but how stem cells maintain quiescence is poorly understood. Here, we show that neural stem cells (NSCs) in the adult mouse hippocampus actively transcribe the pro-activation factor Ascl1 regardless of their activated or quiescent states. We found that the inhibitor of DNA binding protein Id4 is enriched in quiescent NSCs and that elimination of Id4 results in abnormal accumulation of Ascl1 protein and premature stem cell activation. Accordingly, Id4 and other Id proteins promote elimination of Ascl1 protein in NSC cultures. Id4 sequesters Ascl1 heterodimerization partner E47, promoting Ascl1 protein degradation and stem cell quiescence. Our results highlight the importance of non-transcriptional mechanisms for the maintenance of NSC quiescence and reveal a role for Id4 as a quiescence-inducing factor, in contrast with its role of promoting the proliferation of embryonic neural progenitors.

Helix-loop-helix proteins and the advent of cellular diversity: 30 years of discovery.

Helix-loop-helix (HLH) proteins are dimeric transcription factors that control lineage- and developmental-specific gene programs. Genes encoding for HLH proteins arose in unicellular organisms >600 million years ago and then duplicated and diversified from ancestral genes across the metazoan and plant kingdoms to establish multicellularity. Hundreds of HLH proteins have been identified with diverse functions in a wide variety of cell types. HLH proteins orchestrate lineage specification, commitment, self-renewal, proliferation, differentiation, and homing. HLH proteins also regulate circadian clocks, protect against hypoxic stress, promote antigen receptor locus assembly, and program transdifferentiation. HLH proteins deposit or erase epigenetic marks, activate noncoding transcription, and sequester chromatin remodelers across the chromatin landscape to dictate enhancer-promoter communication and somatic recombination. Here the evolution of HLH genes, the structures of HLH domains, and the elaborate activities of HLH proteins in multicellular life are discussed.

ID2 and ID3 are indispensable for Th1 cell differentiation during influenza virus infection in mice.

Antigen-specific Th1 cells could be a passage to the infection sites during infection to execute effector functions, such as help CD8+ T cells to localize in these sites by secretion of anti-viral cytokines-IFN-γ or direct cytotoxicity of antigen-bearing cells. However, the molecular components that modulate Th1 cell differentiation and function in response to viral infection remain incompletely understood. Here, we reported that both inhibitor of DNA binding 3(Id3) protein and inhibitor of DNA binding 2(Id2) protein promoted Th1 cell differentiation. Depletion of Id3 or Id2 led to severe defect of Th1 cell differentiation during influenza virus infection. Whereas depletion of both Id3 and Id2 in CD4+ T cells restrained Th1 cell differentiation to a greater extent, indicating that Id3 and Id2 nonredundantly regulate Th1 cell differentiation. Moreover, deletion of E-proteins, the antagonists of Id proteins, greatly enhanced Th1 cell differentiation. Mechanistic study indicated that E-proteins suppressed Th1 cell differentiation by directly binding to the regulatory elements of Th1 cell master regulator T-bet and regulate T-bet expression. Thus, our findings identified Id-protein's importance for Th1 cells and clarified the nonredundant role of Id3 and Id2 in regulating Th1 cell differentiation, providing novel insight that Id3-Id2-E protein axis are essential for Th1 cell polarization.

E proteins sharpen neurogenesis by modulating proneural bHLH transcription factors' activity in an E-box-dependent manner.

Class II HLH proteins heterodimerize with class I HLH/E proteins to regulate transcription. Here, we show that E proteins sharpen neurogenesis by adjusting the neurogenic strength of the distinct proneural proteins. We find that inhibiting BMP signaling or its target ID2 in the chick embryo spinal cord, impairs the neuronal production from progenitors expressing ATOH1/ASCL1, but less severely that from progenitors expressing NEUROG1/2/PTF1a. We show this context-dependent response to result from the differential modulation of proneural proteins' activity by E proteins. E proteins synergize with proneural proteins when acting on CAGSTG motifs, thereby facilitating the activity of ASCL1/ATOH1 which preferentially bind to such motifs. Conversely, E proteins restrict the neurogenic strength of NEUROG1/2 by directly inhibiting their preferential binding to CADATG motifs. Since we find this mechanism to be conserved in corticogenesis, we propose this differential co-operation of E proteins with proneural proteins as a novel though general feature of their mechanism of action.

Id1 Ablation Protects Hematopoietic Stem Cells from Stress-Induced Exhaustion and Aging.

Defining mechanisms that maintain tissue stem cells during homeostasis, stress, and aging is important for improving tissue regeneration and repair and enhancing cancer therapies. Here, we show that Id1 is induced in hematopoietic stem cells (HSCs) by cytokines that promote HSC proliferation and differentiation, suggesting that it functions in stress hematopoiesis. Genetic ablation of Id1 increases HSC self-renewal in serial bone marrow transplantation (BMT) assays, correlating with decreases in HSC proliferation, mitochondrial biogenesis, and reactive oxygen species (ROS) production. Id1-/- HSCs have a quiescent molecular signature and harbor less DNA damage than control HSCs. Cytokines produced in the hematopoietic microenvironment after γ-irradiation induce Id1 expression. Id1-/- HSCs display a blunted proliferative response to such cytokines and other inducers of chronic proliferation including genotoxic and inflammatory stress and aging, protecting them from chronic stress and exhaustion. Thus, targeting Id1 may be therapeutically useful for improving HSC survival and function during BMT, chronic stress, and aging.

Paradoxical role of Id proteins in regulating tumorigenic potential of lymphoid cells.

A family of transcription factors known as Id proteins, or inhibitor of DNA binding and differentiation, is capable of regulating cell proliferation, survival and differentiation, and is often upregulated in multiple types of tumors. Due to their ability to promote self-renewal, Id proteins have been considered as oncogenes, and potential therapeutic targets in cancer models. On the contrary, certain Id proteins are reported to act as tumor suppressors in the development of Burkitt's lymphoma in humans, and hepatosplenic and innate-like T cell lymphomas in mice. The contexts and mechanisms by which Id proteins can serve in such contradictory roles to determine tumor outcomes are still not well understood. In this review, we explore the roles of Id proteins in lymphocyte development and tumorigenesis, particularly with respect to inhibition of their canonical DNA binding partners known as E proteins. Transcriptional regulation by E proteins, and their antagonism by Id proteins, act as gatekeepers to ensure appropriate lymphocyte development at key checkpoints. We re-examine the derailment of these regulatory mechanisms in lymphocytes that facilitate tumor development. These mechanistic insights can allow better appreciation of the context-dependent roles of Id proteins in cancers and improve considerations for therapy.

The interferon-inducible protein p202 promotes osteogenesis in mouse bone marrow stromal cells.

In the present study, we explored the role of the interferon-inducible protein p202 in osteoblast differentiation of mouse bone marrow stromal cells (BMSCs). Both the mRNA and protein levels of p202 increased initially and decreased afterward in the course of BMSC osteogenesis. The intracellular distribution of this protein also changed in the differentiation process. p202 knockdown inhibited, while p202 overexpression enhanced, the osteoblast differentiation of BMSCs. This was identified by evaluation of expression of osteogenic markers, Alizarin Red S staining, and determination of alkaline phosphatase activity. Further study revealed that p202 disturbs the formation of Runx2/Ids complex and frees Runx2 to induce the differentiation process. The findings demonstrated that p202 plays a positive role in BMSC osteogenesis.