Streamlined discovery of cross-linked chromatin complexes and associated histone modifications by mass spectrometry

Posttranslational modifications (PTMs) are key contributors to chromatin function. The ability to comprehensively link specific histone PTMs with specific chromatin factors would be an important advance in understanding the functions and genomic targeting mechanisms of those factors. We recently introduced a cross-linked affinity technique, BioTAP-XL, to identify chromatin-bound protein interactions that can be difficult to capture with native affinity techniques. However, BioTAP-XL was not strictly compatible with similarly comprehensive analyses of associated histone PTMs. Here we advance BioTAP-XL by demonstrating the ability to quantify histone PTMs linked to specific chromatin factors in parallel with the ability to identify nonhistone binding partners. Furthermore we demonstrate that the initially published quantity of starting material can be scaled down orders of magnitude without loss in proteomic sensitivity. We also integrate hydrophilic interaction chromatography to mitigate detergent carryover and improve liquid chromatography-mass spectrometric performance. In summary, we greatly extend the practicality of BioTAP-XL to enable comprehensive identification of protein complexes and their local chromatin environment.

The Development of Human Megakaryocytes: III. Development of Mature Megakaryocytes From Highly Purified Committed Progenitors in Synthetic Culture Media and Inhibition of Thrombopoietin-Induced Polyploidization by Interleukin-3

Megakaryocyte (MK) progenitors, CD34+CD41+ cells, were isolated from human bone marrow with a purity greater than 98% and a viability of 95%, using affinity techniques with magnetic beads followed by fluorescence-activated cell sorting. These cells were incubated in synthetic media containing the cytokines thrombopoietin (TPO), interleukin-3 (IL-3), stem cell factor (SCF ), and IL-6, obviating the confounding effects of serum growth factors or cytokine secretions of non-MK cells on MK maturation. MK number, MK colony-forming units (CFU-MK), and MK ploidy and phenotype were examined during 7 days in culture. TPO in serum-free cultures without any other exogenously added cytokine supported MK growth and maturation. SCF synergized with TPO to augment MK production and maturation and could partially replace it under some conditions. Both TPO and IL-3 alone increased MK number (12- and 5-fold, respectively) and CFU-MK (∼15-fold each). SCF alone had no effect on MK proliferation in the absence of TPO, but increased both MK number and CFU-MK by 1.5- to 2.0-fold in the presence of TPO. When combined with IL-3, SCF increased both MK number and CFU-MK by 15- to 20-fold in the absence of TPO. In the presence of TPO, the combination of IL-3 and SCF produced only modest increases (1.5- to 2.0-fold) in both MK number and CFU-MK. The proportion of polyploid MK increased greater than fivefold in the presence of TPO. SCF had little effect on MK ploidy in the presence of TPO, but enhanced ploidy twofold to threefold in the absence of TPO. IL-3 alone never increased the level of polyploidization. Rather, it consistently inhibited TPO- and SCF-induced polyploidization of MK. This inhibition was observed in cultures with or without SCF or IL-6. Although IL-3 also supported the proliferation of CD41+ cells and CFU-MK production, the cells that developed under the influence of IL-3 were phenotypically unusual (CD41dim, CD42dim) and of relatively low ploidy. Mature MK were not produced. When added with TPO, IL-3 suppressed polyploidization. Therefore, TPO stimulates MK growth and maturation, whereas IL-3 stimulates growth without maturation and may serve to conserve the immature MK compartment.

The interface between fungal hyphae and orchid protocorm cells

Seeds of the orchids Platanthera hyperborea, Spiranthes lacera, and Spiranthes sinensis were germinated in vitro in the presence of compatible fungal species and the resulting colonized protocorms were studied by light microscopy, transmission electron microscopy, and colloidal-gold affinity techniques. Protocorm cells in early stages of colonization contained coils of fungal hyphae (pelotons) separated from host cell cytoplasm by the host plasma membrane and interfacial matrix material. Host cell walls were labelled by the colloidal gold – cellobiohydralase I (CBH-I) complex to detect cellulose and, particularly over the middle lamella, by antibodies that bind to pectins (JIM 5 and JIM 7). A polyclonal antibody that binds to β-1,3-glucans labelled the fungal cell wall heavily. None of the probes, however, labelled the interfacial matrix between the wall of active fungal hyphae and the surrounding plasma membrane. In contrast, the interfacial matrix material that ensheathed collapsing hyphae showed labelling after treatment with JIM 5, the polyclonal antibody, and the CBH-I complex. Labelling of host cell walls and fungal walls was similar to that described for early stages. Keywords: orchids, protocorms, mycorrhizas, affinity gold techniques, interfacial matrix.