Barrier-to-autointegration Factor Research Articles

Non-random spatial organization of telomeres varies during the cell cycle and requires LAP2 and BAF

Spatial genome organization within the nucleus influences major biological processes and is impacted by the configuration of linear chromosomes. Here, we applied 3D spatial statistics and modeling on high-resolution telomere and centromere 3D-structured illumination microscopy images in cancer cells. We found a multi-scale organization of telomeres that dynamically evolved from a mixed clustered-and-regular distribution in early G1 to a purely regular distribution as cells progressed through the cell cycle. In parallel, our analysis revealed two pools of peripheral and internal telomeres, the proportions of which were inverted during the cell cycle. We then conducted a targeted screen using MadID to identify the molecular pathways driving or maintaining telomere anchoring to the nuclear envelope observed in early G1. Lamina-associated polypeptide (LAP) proteins were found transiently localized to telomeres in anaphase, a stage where LAP2α initiates the reformation of the nuclear envelope, and impacted telomere redistribution in the next interphase together with their partner barrier-to-autointegration factor (BAF).

Special Nuclear Structures in the Germinal Vesicle of the Common Frog with Emphasis on the So-Called Karyosphere Capsule.

The karyosphere (karyosome) is a structure that forms in the oocyte nucleus-germinal vesicle (GV)-at the diplotene stage of meiotic prophase due to the assembly of all chromosomes in a limited portion of the GV. In some organisms, the karyosphere has an extrachromosomal external capsule, the marker protein of which is nuclear F-actin. Despite many years of theories about the formation of the karyosphere capsule (KC) in the GV of the common frog Rana temporaria, we present data that cast doubt on its existence, at least in this species. Specific extrachromosomal strands, which had been considered the main elements of the frog's KC, do not form a continuous layer around the karyosphere and, according to immunogold labeling, do not contain structural proteins, such as actin and lamin B. At the same time, F-actin is indeed noticeably concentrated around the karyosphere, creating the illusion of a capsule at the light microscopy/fluorescence level. The barrier-to-autointegration factor (BAF) and one of its functional partners-LEMD2, an inner nuclear membrane protein-are not localized in the strands, suggesting that the strands are not functional counterparts of the nuclear envelope. The presence of characteristic strands in the GV of R. temporaria late oocytes may reflect an excess of SMC1 involved in the structural maintenance of diplotene oocyte chromosomes at the karyosphere stage, since SMC1 has been shown to be the most abundant protein in the strands. Other characteristic microstructures-the so-called annuli, very similar in ultrastructure to the nuclear pore complexes-do not contain nucleoporins Nup35 and Nup93, and, therefore, they cannot be considered autonomous pore complexes, as previously thought. Taken together, our data indicate that traditional ideas about the existence of the R. temporaria KC as a special structural compartment of the GV are to be revisited.

Identification of PP2A-B55 targets uncovers regulation of emerin during nuclear envelope reassembly in Drosophila.

Mitotic exit requires the dephosphorylation of many proteins whose phosphorylation was needed for mitosis. Protein phosphatase 2A with its B55 regulatory subunit (PP2A-B55) promotes this transition. However, the events and substrates that it regulates are incompletely understood. We used proteomic approaches in Drosophila to identify proteins that interact with and are dephosphorylated by PP2A-B55. Among several candidates, we identified emerin (otefin in Drosophila). Emerin resides in the inner nuclear membrane and interacts with the DNA-binding protein barrier-to-autointegration factor (BAF) via a LEM domain. We found that the phosphorylation of emerin at Ser50 and Ser54 near its LEM domain negatively regulates its association with BAF, lamin and additional emerin in mitosis. We show that dephosphorylation of emerin at these sites by PP2A-B55 determines the timing of nuclear envelope reformation. Genetic experiments indicate that this regulation is required during embryonic development. Phosphoregulation of the emerin-BAF complex formation by PP2A-B55 appears as a key event of mitotic exit that is likely conserved across species.

A De Novo Sequence Variant in Barrier-to-Autointegration Factor Is Associated with Dominant Motor Neuronopathy.

Barrier-to-autointegration factor (BAF) is an essential component of the nuclear lamina. Encoded by BANF1, this DNA binding protein contributes to the regulation of gene expression, cell cycle progression, and nuclear integrity. A rare recessive BAF variant, Ala12Thr, causes the premature aging syndrome, Néstor-Guillermo progeria syndrome (NGPS). Here, we report the first dominant pathogenic BAF variant, Gly16Arg, identified in a patient presenting with progressive neuromuscular weakness. Although disease variants carry nearby amino acid substitutions, cellular and biochemical properties are distinct. In contrast to NGPS, Gly16Arg patient fibroblasts show modest changes in nuclear lamina structure and increases in repressive marks associated with heterochromatin. Structural studies reveal that the Gly16Arg substitution introduces a salt bridge between BAF monomers, reducing the conformation ensemble available to BAF. We show that this structural change increases the double-stranded DNA binding affinity of BAF Gly16Arg. Together, our findings suggest that BAF Gly16Arg has an increased chromatin occupancy that leads to epigenetic changes and impacts nuclear functions. These observations provide a new example of how a missense mutation can change a protein conformational equilibrium to cause a dominant disease and extend our understanding of mechanisms by which BAF function impacts human health.

Mechanistic Insights of the LEMD2 p.L13R Mutation and Its Role in Cardiomyopathy.

Nuclear envelope proteins play an important role in the pathogenesis of hereditary cardiomyopathies. Recently, a new form of arrhythmic cardiomyopathy caused by a homozygous mutation (p.L13R) in the inner nuclear membrane protein LEMD2 was discovered. The aim was to unravel the molecular mechanisms of mutant LEMD2 in the pathogenesis of cardiomyopathy. We generated a Lemd2 p.L13R knock-in mouse model and a corresponding cell model via CRISPR/Cas9 technology and investigated the cardiac phenotype as well as cellular and subcellular mechanisms of nuclear membrane rupture and repair. Knock-in mice developed a cardiomyopathy with predominantly endocardial fibrosis, left ventricular dilatation, and systolic dysfunction. Electrocardiograms displayed pronounced ventricular arrhythmias and conduction disease. A key finding of knock-in cardiomyocytes on ultrastructural level was a significant increase in nuclear membrane invaginations and decreased nuclear circularity. Furthermore, increased DNA damage and premature senescence were detected as the underlying cause of fibrotic and inflammatory remodeling. As the p.L13R mutation is located in the Lap2/Emerin/Man1 (LEM)-domain, we observed a disrupted interaction between mutant LEMD2 and BAF (barrier-to-autointegration factor), which is required to initiate the nuclear envelope rupture repair process. To mimic increased mechanical stress with subsequent nuclear envelope ruptures, we investigated mutant HeLa-cells upon electrical stimulation and increased stiffness. Here, we demonstrated impaired nuclear envelope rupture repair capacity, subsequent cytoplasmic leakage of the DNA repair factor KU80 along with increased DNA damage, and recruitment of the cGAS (cyclic GMP-AMP synthase) to the nuclear membrane and micronuclei. We show for the first time that the Lemd2 p.L13R mutation in mice recapitulates human dilated cardiomyopathy with fibrosis and severe ventricular arrhythmias. Impaired nuclear envelope rupture repair capacity resulted in increased DNA damage and activation of the cGAS/STING/IFN pathway, promoting premature senescence. Hence, LEMD2 is a new player inthe disease group of laminopathies.

Immunohistochemical Analysis of Nuclear Lamina Structures in the Drosophila Ovary Using CRISPR-Tagged Genes.

The Drosophila ovary represents an outstanding model for investigating tissue homeostasis. Females continuously produce oocytes throughout their lifetime. However, as females age, fecundity declines, in part, due to changes in ovarian niche function and germline stem cell (GSC) homeostasis. Understanding the dynamics of GSC maintenance will provide needed insights into how coordinated tissue homeostasis is lost during aging. Critical regulators of GSC maintenance are proteins that reside in the nuclear lamina (NL), including the NL proteins emerin and Barrier-to-Autointegration Factor (BAF). Continued investigation of how emerin, BAF, and other NL proteins contribute to GSC function depends upon the availability of antibodies for NL proteins, a limiting resource. In this chapter, we discuss strategies for using clustered regularly interspaced short palindromic repeats (CRISPR) genomic editing to produce endogenously tagged NL genes to circumvent this obstacle, using the generation of the gfp-baf allele as an example. We describe strategies for validation of tagged alleles. Finally, we outline methods for immunohistochemical analysis of resulting tagged-NL proteins.

The Conformation of the Intrinsically Disordered N-Terminal Region of Barrier-to-Autointegration Factor (BAF) is Regulated by pH and Phosphorylation

Barrier-to-Autointegration Factor (BAF) is a highly conserved DNA binding protein important for genome integrity. Its localization and function are regulated through phosphorylation. Previously reported structures of BAF suggested that it is fully ordered, but our recent NMR analysis revealed that its N-terminal region is flexible in solution and that S4/T3 di-phosphorylation by VRK1 reduces this flexibility. Here, molecular dynamics (MD) simulation was used to unveil the conformational ensembles accessible to the N-terminal region of BAF either unphosphorylated, mono-phosphorylated on S4 or di-phosphorylated on S4/T3 (pBAF) and to reveal the interactions that contribute to define these ensembles. We show that the intrinsic flexibility observed in the N-terminal region of BAF is reduced by S4 phosphorylation and to a larger extent by S4/T3 di-phosphorylation. Thanks to the atomic description offered by MD supported by the NMR study of several BAF mutants, we identified the dynamic network of salt bridge interactions responsible for the conformational restriction involving pS4 and pT3 with residues located in helix α1 and α6. Using MD, we showed that the flexibility in the N-terminal region of BAF depends on the ionic strength and on the pH. We show that the presence of two negative charges of the phosphoryl groups is required for a substantial decrease in flexibility in pBAF. Using MD supported by NMR, we also showed that H7 deprotonation reduces the flexibility in the N-terminal region of BAF. Thus, the conformation of the intrinsically disordered N-terminal region of BAF is highly tunable, likely related to its diverse functions.

CNSC-36. VRK1 IS A PARALOG SYNTHETIC LETHAL TARGET IN VRK2-METHYLATED GLIOBLASTOMA

Abstract Synthetic lethality — a genetic interaction that results in cell death when two genetic deficiencies co-occur but not when either deficiency occurs alone — can be co-opted for cancer therapeutics. A pair of paralog genes is among the most straightforward synthetic lethal interaction by virtue of their redundant functions. Here we demonstrate a paralog-based synthetic lethality by targeting Vaccinia-Related Kinase 1 (VRK1) in Vaccinia-Related Kinase 2 (VRK2)-methylated glioblastoma (GBM). VRK2 is silenced by promoter methylation in approximately two-thirds of GBM, an aggressive cancer with few available targeted therapies. Genetic knockdown of VRK1 in VRK2-methylated GBM cell lines and patient-derived models was lethal and resulted in decreased activity of the downstream substrate Barrier to Autointegration Factor (BAF), a regulator of post-mitotic nuclear envelope formation. VRK1 knockdown, and thus reduced BAF activity, caused nuclear lobulation, blebbing and micronucleation, which subsequently resulted in G2/M arrest and DNA damage. The VRK1-VRK2 synthetic lethal interaction was dependent on VRK1 kinase activity and was rescued by ectopic VRK2 expression. Knockdown of VRK1 led to robust tumor growth inhibition in VRK2-methylated GBM xenografts. These results indicate that inhibiting VRK1 kinase activity could be a viable therapeutic strategy in VRK2-methylated GBM.

Nucleoplasmic lamin C rapidly accumulates at sites of nuclear envelope rupture with BAF and cGAS.

In mammalian cell nuclei, the nuclear lamina (NL) underlies the nuclear envelope (NE) to maintain nuclear structure. The nuclear lamins, the major structural components of the NL, are involved in the protection against NE rupture induced by mechanical stress. However, the specific role of the lamins in repair of NE ruptures has not been fully determined. Our analyses using immunofluorescence and live-cell imaging revealed that the nucleoplasmic pool of lamin C rapidly accumulated at sites of NE rupture induced by laser microirradiation in mouse embryonic fibroblasts. The accumulation of lamin C at the rupture sites required both the immunoglobulin-like fold domain that binds to barrier-to-autointegration factor (BAF) and a nuclear localization signal. The accumulation of nuclear BAF and cytoplasmic cyclic GMP-AMP synthase (cGAS) at the rupture sites was in part dependent on lamin A/C. These results suggest that nucleoplasmic lamin C, BAF, and cGAS concertedly accumulate at sites of NE rupture for rapid repair.

VRK1 Is a Synthetic-Lethal Target in VRK2-Deficient Glioblastoma.

A paralog synthetic-lethal interaction between VRK1 and VRK2 sensitizes VRK2-methylated glioblastoma to perturbation of VRK1 kinase activity, supporting VRK1 as a drug discovery target in this disease.

The BAF A12T mutation disrupts lamin A/C interaction, impairing robust repair of nuclear envelope ruptures in Nestor-Guillermo progeria syndrome cells.

Nestor–Guillermo progeria syndrome (NGPS) is caused by a homozygous alanine-to-threonine mutation at position 12 (A12T) in barrier-to-autointegration factor (BAF). It is characterized by accelerated aging with severe skeletal abnormalities. BAF is an essential protein binding to DNA and nuclear envelope (NE) proteins, involved in NE rupture repair. Here, we assessed the impact of BAF A12T on NE integrity using NGPS-derived patient fibroblasts. We observed a strong defect in lamin A/C accumulation to NE ruptures in NGPS cells, restored upon homozygous reversion of the pathogenic BAF A12T mutation with CRISPR/Cas9. By combining in vitro and cellular assays, we demonstrated that while the A12T mutation does not affect BAF 3D structure and phosphorylation by VRK1, it specifically decreases the interaction between BAF and lamin A/C. Finally, we revealed that the disrupted interaction does not prevent repair of NE ruptures but instead generates weak points in the NE that lead to a higher frequency of NE re-rupturing in NGPS cells. We propose that this NE fragility could directly contribute to the premature aging phenotype in patients.

Ankle2 deficiency-associated microcephaly and spermatogenesis defects in zebrafish are alleviated by heterozygous deletion of vrk1

Autosomal recessive primary microcephaly (MCPH) is a rare congenital disorder characterized by a below average brain volume at birth and is associated with neurodevelopmental disorders such as growth retardation and intellectual disability. Mutations in ANKLE2 have been identified as one of the causes of MCPH (MCPH16). ANKLE2 is a target molecule of the Zika virus NS4a protein that interferes with ANKLE2 function, resulting in severe microcephaly. ANKLE2 is essential for organizing the nuclear envelope and chromatin structures during the mitotic-end process via barrier to autointegration factor (BAF) dephosphorylation. However, the precise mechanism by which the loss of ANKLE2 function causes the pathogenesis of microcephaly remains unclear. In this study, we generated Ankle2-deficient zebrafish (ankle2−/−) with a significant reduction in brain size compared with that of their control siblings. The ankle2−/− brain showed a significant decrease in the number of radial glial progenitor cells, suggesting that Ankle2 deficiency in zebrafish causes neurogenesis defects. Furthermore, ankle2−/− male zebrafish showed infertility owing to defects in spermatogenesis. Notably, microcephaly was overcome by vrk1 morpholino knockdown or vrk1 heterozygous deletion. In addition, spermatogenesis in ankle2−/− zebrafish males was partially restored by the vrk1 heterozygous deletion, although infertility was not resolved. These results indicate that ANKLE2 and VRK1 coordinate with each other for BAF phosphorylation to maintain normal mitosis during neurogenesis and spermatogenesis.

Checkpoint activation drives global gene expression changes in Drosophila nuclear lamina mutants.

The nuclear lamina (NL) lines the inner nuclear membrane. This extensive protein network organizes chromatin and contributes to the regulation of transcription, DNA replication, and repair. Lap2-emerin-MAN1 domain (LEM-D) proteins are key members of the NL, representing proteins that connect the NL to the genome through shared interactions with the chromatin-binding protein Barrier-to-Autointegration Factor (BAF). Functions of the LEM-D protein emerin and BAF are essential during Drosophila melanogaster oogenesis. Indeed, loss of either emerin or BAF blocks germ cell development and causes loss of germline stem cells, defects linked to the deformation of NL structure, and non-canonical activation of Checkpoint kinase 2 (Chk2). Here, we investigate the contributions of emerin and BAF to gene expression in the ovary. Profiling RNAs from emerin and baf mutant ovaries revealed that nearly all baf misregulated genes were shared with emerin mutants, defining a set of NL-regulated genes. Strikingly, loss of Chk2 restored the expression of most NL-regulated genes, identifying a large class of Chk2-dependent genes (CDGs). Nonetheless, some genes remained misexpressed upon Chk2 loss, identifying a smaller class of emerin-dependent genes (EDGs). Properties of EDGs suggest a shared role for emerin and BAF in the repression of developmental genes. Properties of CDGs demonstrate that Chk2 activation drives global misexpression of genes in the emerin and baf mutant backgrounds. Notably, CDGs were found upregulated in lamin-B mutant backgrounds. These observations predict that Chk2 activation might have a general role in gene expression changes found in NL-associated diseases, such as laminopathies.

Mus musculus Barrier-To-Autointegration Factor 2 (Banf2) is Not Essential for Spermatogenesis or Fertility

The barrier-to-autointegration factor (BAF) is widely expressed in most human tissues and plays a critical role in chromatin organization, nuclear envelope assembly, gonadal development, and embryonic stem cell self-renewal. Complete loss of BAF has been shown to lead to embryonic lethality and gonadal defects. The BAF paralog, namely, barrier-to-autointegration factor 2 (BANF2), exhibits a testis-predominant expression pattern in both humans and mice. Unlike BAF, it may cause isolated male infertility. Therefore, we used the CRISPR/Cas9 system to generate Banf2-knockout mice to further study its function in spermatogenesis. Unexpectedly, knockout mice did not show any detectable abnormalities in histological structure of the testis, epididymis, ovary, and other tissues, and exhibited normal fertility, indicating that Banf2 is not essential for mouse spermatogenesis and fertility.

Development of a structure-analysis pipeline using multiple-solvent crystal structures of barrier-to-autointegration factor.

The multiple-solvent crystal structure (MSCS) approach uses high concentrations of organic solvents to characterize the interactions and effects of solvents on proteins. Here, the method has been further developed and an MSCS data-handling pipeline is presented that uses the Detection of Related Solvent Positions (DRoP) program to improve data quality. DRoP is used to selectively model conserved water molecules, so that an advanced stage of structural refinement is reached quickly. This allows the placement of organic molecules more accurately and convergence on high-quality maps and structures. This pipeline was applied to the chromatin-associated protein barrier-to-autointegration factor (BAF), resulting in structural models with better than average statistics. DRoP and Phenix Structure Comparison were used to characterize the data sets and to identify a binding site that overlaps with the interaction site of BAF with emerin. The conserved water-mediated networks identified by DRoP suggested a mechanism by which water molecules are used to drive the binding of DNA. Normalized and differential B-factor analysis is shown to be a valuable tool to characterize the effects of specific solvents on defined regions of BAF. Specific solvents are identified that cause stabilization of functionally important regions of the protein. This work presents tools and a standardized approach for the analysis and comprehension of MSCS data sets.

Protein structural and mechanistic basis of progeroid laminopathies.

Progeroid laminopathies are characterized by the premature appearance of certain signs of physiological aging in a subset of tissues. They are caused by mutations in genes coding for A-type lamins or lamin-binding proteins. Here, we review how different mutations causing progeroid laminopathies alter protein structure or protein-protein interactions and how these impact on mechanisms that protect cell viability and function. One group of progeroid laminopathies, which includes Hutchinson-Gilford progeria syndrome, is characterized by accumulation of unprocessed prelamin A or variants. These are caused by mutations in the A-type lamin gene (LMNA), altering prelamin A itself, or in ZMPSTE24, encoding an endoprotease involved in its processing. The abnormally expressed farnesylated proteins impact on various cellular processes that may contribute to progeroid phenotypes. Other LMNA mutations lead to the production of nonfarnesylated A-type lamin variants with amino acid substitutions in solvent-exposed hot spots located mainly in coil 1B and the immunoglobulin fold domain. Dominant missense mutations might reinforce interactions between lamin domains, thus giving rise to excessively stabilized filament networks. Recessive missense mutations in A-type lamins and barrier-to-autointegration factor (BAF) causing progeroid disorders are found at the interface between these interacting proteins. The amino acid changes decrease the binding affinity of A-type lamins for BAF, which may contribute to lamina disorganization, as well as defective repair of mechanically induced nuclear envelope rupture. Targeting these molecular alterations in A-type lamins and associated proteins identified through structural biology studies could facilitate the design of therapeutic strategies to treat patients with rare but severe progeroid laminopathies.

A fraction of barrier-to-autointegration factor (BAF) associates with centromeres and controls mitosis progression

Barrier-to-Autointegration Factor (BAF) is a conserved nuclear envelope (NE) component that binds chromatin and helps its anchoring to the NE. Cycles of phosphorylation and dephosphorylation control BAF function. Entering mitosis, phosphorylation releases BAF from chromatin and facilitates NE-disassembly. At mitotic exit, PP2A-mediated dephosphorylation restores chromatin binding and nucleates NE-reassembly. Here, we show that in Drosophila a small fraction of BAF (cenBAF) associates with centromeres. We also find that PP4 phosphatase, which is recruited to centromeres by CENP-C, prevents phosphorylation and release of cenBAF during mitosis. cenBAF is necessary for proper centromere assembly and accurate chromosome segregation, being critical for mitosis progression. Disrupting cenBAF localization prevents PP2A inactivation in mitosis compromising global BAF phosphorylation, which in turn leads to its persistent association with chromatin, delays anaphase onset and causes NE defects. These results suggest that, together with PP4 and CENP-C, cenBAF forms a centromere-based mechanism that controls chromosome segregation and mitosis progression.

DNA unchained: two assays to discover and study inhibitors of the DNA clustering function of barrier-to-autointegration factor

The protein barrier-to-autointegration factor (BAF) and its interaction partners, the LEM (LAP2B, emerin, MAN1)-domain proteins, constitute a powerful cytoplasmic DNA defense mechanism. Invading DNA molecules are quickly bound by the BAF system and trapped in membrane compartments. This decreases the nuclear uptake of DNA from the cytoplasm. Inhibition of the BAF system is therefore expected to enhance the efficacy of non-viral DNA transfection agents. In this study, we introduced a protocol for the recombinant expression of soluble BAF and developed two ELISA-type assays to discover small molecule inhibitors of BAF-dependent DNA retention by high throughput screening (HTS). The proton pump inhibitor rabeprazole as well as three compounds of the Maybridge library were identified as inhibitors of the LEM-BAF-DNA interaction chain. The inhibition was based on adduct formation with BAF cysteine residues. An enhancing effect of the compounds on cell culture transfection, however, was not observed, which may be attributed to the reducing environment of the cytoplasm that prevents the adduct formation with BAF cysteine residues. The novel assays developed here can provide new tools to further study the biological functions of the BAF system, and may lead to the identification of suitable BAF inhibitors in future HTS campaigns.

A barrier-to-autointegration factor promotes white spot syndrome virus infection in a crustacean Cherax quadricarinatus

Barrier-to-autointegration factor (BAF) is a highly conserved DNA binding protein that participates in a variety of biological processes such as transcription, epigenetic regulation and antiviral immunity in vertebrates. However, the function of BAF is poorly understood in crustaceans. In this study, we identified a barrier-to-autointegration factor (CqBAF) from red claw crayfish Cherax quadricarinatus, which was responsive to white spot syndrome virus (WSSV) infection. The full-length cDNA sequence of CqBAF was 544 bp, including an open reading frame of 273 bp encoding 90 amino acids, a 107 bp of 5′-Untranslated Regions (5′-UTR) and a 164 bp of 3′-UTR. Gene expression analysis showed that CqBAF was distributed in all tissues examined with the highest expression in the crayfish haematopietic tissue (Hpt), which protein expression was also significantly up-regulated by WSSV infection in Hpt cells. Furthermore, the transcripts of both an immediate early gene IE1 and a late envelope protein gene VP28 of WSSV were clearly reduced in Hpt cells after gene silencing of CqBAF. Importantly, the promoter activity of two immediate early genes of WSSV, including WSV051 and IE1, was strongly enhanced by the increased phosphorylation of CqBAF, which also facilitated the accumulation of CqBAF protein in the cytoplasm of Sf9 cells. Taken together, these data suggest that CqBAF is likely to increase the replication of WSSV by promoting the transcription of viral immediate early genes, probably regulated by phosphorylation of CqBAF, which sheds new light on the molecular mechanism of WSSV infection.

Survival of Drosophila germline stem cells requires the chromatin-binding protein Barrier-to-autointegration factor.

The nuclear lamina (NL) is an extensive protein network that underlies the inner nuclear envelope. This network includes LAP2-emerin-MAN1 domain (LEM-D) proteins that associate with the chromatin and DNA-binding protein Barrier-to-autointegration factor (BAF). Here, we investigate the partnership between three NL Drosophila LEM-D proteins and BAF. In most tissues, only Emerin/Otefin is required for NL enrichment of BAF, revealing an unexpected dependence on a single LEM-D protein. Prompted by these observations, we studied BAF contributions in the ovary, a tissue where Emerin/Otefin function is essential. We show that germ cell-specific BAF knockdown causes phenotypes that mirror emerin/otefin mutants. Loss of BAF disrupts NL structure, blocks differentiation and promotes germ cell loss, phenotypes that are partially rescued by inactivation of the ATR and Chk2 kinases. These data suggest that, similar to emerin/otefin mutants, BAF depletion activates the NL checkpoint that causes germ cell loss. Taken together, our findings provide evidence for a prominent NL partnership between the LEM-D protein Emerin/Otefin and BAF, revealing that BAF functions with this partner in the maintenance of an adult stem cell population.