Legant, W. | bioRxiv Science

bioRxiv2026

Aberrant chromatin looping by NUP98-HOXA9 is constrained by CTCF and facilitated by cohesin

The acute myeloid leukemia (AML) fusion protein NUP98-HOXA9 (NHA9) drives leukemogenesis by promoting aberrant chromatin loop formation through phase separation, yet the mechanisms underlying these interactions remain unclear. To address this, we dissect the interplay between NHA9 and individual loop extrusion factors using in situ Hi-C, CUT&RUN, RNA-seq, and Auxin-inducible degradation of CTCF or RAD21. CTCF was found to be dispensable for NHA9 loop formation, although CTCF binding constrained a subset of loops that emerged only upon CTCF depletion. In contrast, cohesin played a distance-dependent role where short-range NHA9 loops formed independently of RAD21, while long-range loops were strongly cohesin-dependent. Despite this requirement, RAD21 showed minimal enrichment at NHA9 loop anchors, indicating that NHA9 does not function as a canonical cohesin barrier. Instead, these findings support a non-canonical model in which cohesin transiently facilitates interactions between distal NHA9-bound loci, which are subsequently stabilized through NHA9 phase separation. Together, this work reveals a distinct mechanism of oncogenic chromatin looping in which NUP98-HOXA9 cooperates with canonical loop extrusion machinery to reprogram genome architecture in AML.

Molecular Biology↗

bioRxiv2024

Measurement of cellular traction forces during confined migration

To migrate efficiently through tissues, cells must transit through small constrictions within the extracellular matrix. However, in vivo environments are geometrically, mechanically, and chemically complex, and it has been difficult to understand how each of these parameters contribute to the propulsive strategy utilized by cells in these diverse settings. To address this, we employed a sacrificial micromolding approach to generate polymer substrates with tunable stiffness, controlled adhesivity, and user-defined microscale geometries. We combined this together with live-cell imaging and three-dimensional traction force microscopy (TFM) to quantify the forces that cells use to transit through constricting channels. Surprisingly, we observe that cells migrating through compliant constrictions take longer to transit and experience greater nuclear deformation than those migrating through more rigid constrictions. TFM reveals that this deformation is generated by inwardly directed contractile forces that decrease the size of the opening and pull the walls closed around the nucleus. These findings show that nuclear deformation during confined migration can be accomplished by internal cytoskeletal machinery rather than by reactive forces from the substrate, and our approach provides a mechanism to test between different models for how cells translocate their nucleus through narrow constrictions. The methods, analysis, and results presented here will be useful to understand how cells choose between propulsive strategies in different physical environments. Significance StatementCell migration is critical for both physiological events like wound healing and pathological events like metastasis. Understanding how cells move through complex environments will assist efforts to enhance or inhibit such processes. We developed a method to quantify the forces that cells use to move through multidimensional environments, including through narrow constrictions like those in tissues. Surprisingly, we find that cells transiting through soft constrictions take longer and deform more than those transiting through rigid constrictions, and we connect this finding to inwardly directed contractile forces generated by migrating cells. Together, this work reveals a key role for substrate rigidity to regulate cell transit through confining geometries and provides a quantitative platform to investigate similar processes in other settings.

biophysics↗